Antibodies that bind alphaE integrin

ABSTRACT

Antibodies and antigen-binding fragments of antibodies that bind αE integrin are disclosed. Some of the antibodies and antigen-binding fragments bind an activation induced epitope on integrin αE chain. In some embodiments, the antibodies are human. Nucleic acids and vectors encoding the antibodies or portions thereof, recombinant cells that contain the nucleic acids, and compositions comprising the antibodies or antigen-binding fragments are also disclosed. The invention also provides therapeutic and diagnostic methods that employ the antibodies and antigen-binding fragments.

BACKGROUND OF THE INVENTION

[0001] Integrin receptors are important for regulating both lymphocyterecirculation and recruitment to sites of inflammation (Carlos, T. M.and Harlan, J. M., Blood 84:2068-2101 (1994)). The αE integrin αEβ7 isexpressed on mucosal homing lymphocytes such as intestinalintraepithelial lymphocytes (IEL) and binds E-cadherin, which isexpressed on epithelial cells, as well as a ligand on intestinalmicrovascular endothelial cell lines (Cepek, K. L. et al., Nature372:190-193 (1994); Stauch U. G. et al., J. Immunol. 166:3506-3514(2001)). As such, the αEβ7 integrin acts as a homing receptor thatmediates lymphocyte migration to mucosal epithelium, such as intestinalepithelium (Schon, M. P. et al., J. Immunol. 162:6641-6649 (1999)).

[0002] αE integrins, like other integrins, can assume an activated orinactive conformation. Activated integrins bind ligand (e.g. E-cadherin)with high affinity. αE integrins, such as αEβ7, can be activated bydivalent cations and/or by inside out signalling upon cellularstimulation with mitogens, growth factors and/or specific antigen (e.g.,peptide/MHC).

[0003] Antibodies which bind αEβ7 integrin can interfere with αEβ7integrin binding to its ligands (e.g., E-cadherin) and inhibit leukocytemigration to mucosal inflammatory sites (see, e.g., Ludviksson, B. R. etal., J. Immunol. 162:4975-4982 (1999); WO 00/30681 (Ludviksson, B. R. etal.)). However, a problem with using murine antibodies or othernon-human antibodies for in vivo applications (e.g., diagnostic methods,therapeutic methods) in humans is that they are highly immunogenic andquickly induce a human anti-foreign antibody response (e.g., a humananti-mouse antibody response, HAMA). Such a human anti-foreign antibodyresponse can result in rapid clearance of the foreign antibody andseverely limit diagnostic or therapeutic uses or abrogate anytherapeutic benefits.

[0004] Thus, a need exists for improved antibodies and antigen-bindingfragments that can be used to diagnose and/or treat subjects havingmucosal inflammatory disorders.

SUMMARY OF THE INVENTION

[0005] The invention relates to antibodies and antigen-binding fragmentsof antibodies which bind an αE integrin (e.g., αEβ7 or other integrincomprising an αE chain). In one aspect, the invention is an antibody orantigen-binding fragment thereof that binds an activation-inducedepitope on integrin αE chain, such as an epitope induced by exposure ofan αE integrin to a divalent cation (e.g., Mn²⁺). For example, theactivation-induced epitope can comprise amino acid residues in the Idomain of integrin αE chain. In one embodiment, the antibody orantigen-binding fragment thereof binds an activation-induced epitope onhuman integrin αE chain. In another embodiment, the antibody orantigen-binding fragment thereof can inhibit the binding of a ligand(e.g., E-cadherin) to an αE integrin (e.g., αEβ7). In other embodiments,the antibody or antigen-binding fragment can inhibit αEintegrin-mediated adhesion of a first cell expressing an αE integrin toa second cell bearing a ligand of an αE integrin, such as epithelialcells (e.g., intestinal epithelial cells) or endothelial cells. Inparticular embodiments, the antibody or antigen-binding fragmentcompetitively inhibits binding of mAb 3G6 to αEβ7 integrin, or has theepitopic specificity of mAb 3G6.

[0006] In other embodiments, the antibody comprises one, two or threeheavy chain complementarity determining regions (HCDR1, HCDR2 and/orHCDR3) having the amino acid sequences of the heavy chain CDRs of mAb3G6 wherein, optionally, one or two amino acids in each heavy chain CDRcan be conservatively substituted, and one, two or three light chaincomplementarity determining regions (LCDR1, LCDR2 and/or LCDR3) havingthe amino acid sequences of the light chain CDRs of mAb 3G6 wherein,optionally, one or two amino acids in each light chain CDR can beconservatively substituted. Preferably, the antibody comprises the threeheavy chain CDRs and the three light chain CDRs of mAb 3G6. For example,in a particular embodiment the antibody can comprise the heavy chainvariable region of mAb 3G6 (SEQ ID NO: 4) and the light chain variableregion of mAb 3G6 (SEQ ID NO: 9).

[0007] In other embodiments, the antibody comprises one, two or threeheavy chain complementarity determining regions (HCDR1, HCDR2 and/orHCDR3) having the amino acid sequences of the heavy chain CDRs of mAb5E4 wherein, optionally, one or two amino acids in each heavy chain CDRcan be conservatively substituted, and one, two or three light chaincomplementarity determining regions (LCDR1, LCDR2 and/or LCDR3) havingthe amino acid sequences of the light chain CDRs of mAb 5E4 wherein,optionally, one or two amino acids in each light chain CDR can beconservatively substituted. Preferably, the antibody comprises the threeheavy chain CDRs and the three light chain CDRs of mAb 5E4. For example,in a particular embodiment the antibody can comprise the heavy chainvariable region of mAb 5E4 (SEQ ID NO: 14) and the light chain variableregion of mAb 5E4 (SEQ ID NO: 19).

[0008] In additional embodiments, the antibody comprises one, two orthree heavy chain complementarity determining regions (HCDR1, HCDR2and/or HCDR3) having the amino acid sequences of the heavy chain CDRs ofmAb 8D5 wherein, optionally, one or two amino acids in each heavy chainCDR can be conservatively substituted, and one, two or three light chaincomplementarity determining regions (LCDR1, LCDR2 and/or LCDR3) havingthe amino acid sequences of the light chain CDRs of mAb 8D5 wherein,optionally, one or two amino acids in each light chain CDR can beconservatively substituted. Preferably, the antibody comprises the threeheavy chain CDRs and the three light chain CDRs of mAb 8D5. For example,in a particular embodiment the antibody can comprise the heavy chainvariable region of mAb 8D5 (SEQ ID NO: 24) and the light chain variableregion of mAb 8D5 (SEQ ID NO: 29).

[0009] Preferred antibodies that bind an αE integrin (e.g., selectivelybind an activation-induced epitope on integrin αE chain) includechimeric antibodies, humanized antibodies and antigen-binding fragmentsof the foregoing. Particularly preferred antibodies are of human origin.In specific embodiments, the invention is mAb 3G6, mAb 5E4 or mAb 8D5 oran antigen-binding fragment of mAb 3G6, mAb 5E4 or mAb 8D5.

[0010] The invention also relates to the heavy chains, light chains andportions of the heavy chains and light chains of the antibodiesdescribed herein. The invention also relates to fusion proteinscomprising an antibody or portion thereof (e.g., heavy chain, lightchain, variable region) of the invention and a non-immunoglobulinmoiety. The invention also relates to immuno-conjugates comprising anantibody or antigen-binding fragment of the invention and a secondmoiety, such as a toxin (e.g., cytotoxin, cytotoxic agent), atherapeutic agent (e.g., a chemotherapeutic agent, an antimetabolite, analkylating agent, an anthracycline, an antibiotic, an anti-mitoticagent, a biological response modifier (e.g., a cytokine (e.g., aninterleukin, an interferon, a tumor necrosis factor), a growth factor(e.g., a neurotrophic factor)), a plasminogen activator, a radionuclide(e.g, a radioactive ion) or enzyme, for example.

[0011] The invention also relates to isolated and/or recombinant nucleicacids encoding the antibodies, antigen-binding fragments, heavy chains,light chains and portions of the heavy chains and light chains of theantibodies described herein, and to expression constructs or vectorscomprising same. The invention also relates to a host cell thatcomprises a nucleic acid of the invention. In specific embodiments, theinvention is hybridoma 3G6, hybridoma 5E4 or hybridoma 8D5.

[0012] The invention also relates to a method of treating a subjecthaving an inflammatory disease or disorder comprising administering tosaid subject an effective amount of an antibody or antigen-bindingfragment of the invention. In particular embodiments, the subject is ahuman. In other particular embodiments, the subject has an inflammatorybowel disease, such as ulcerative colitis or Crohn's disease.

[0013] The invention also relates to a method for detecting an activatedαE integrin (e.g., activated αEβ7) comprising contacting a compositioncomprising an αE integrin with an antibody or antigen-binding fragmentthereof which binds an activation-induced epitope on integrin αE chainand detecting formation of a complex between said antibody orantigen-binding fragment and said activated αE integrin.

[0014] The invention further relates to an antibody, antigen-bindingfragment of an antibody, fusion protein or immuno-conjugate as describedherein for use in therapy (including prophylaxis) or diagnosis, and tothe use of an antibody, antigen-binding fragment of an antibody, fusionprotein or immuno-conjugate of the invention for the manufacture of amedicament for the treatment of a particular disease or condition asdescribed herein (e.g., a mucosal inflammatory disease (e.g.,inflammatory bowel disease (e.g., ulerative colitis, Crohn's disease)),cancer (e.g., leukemia, lymphoma)).

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1H are fluorescent histograms showing binding of mAb 3G6(IgG1) to transfected K562 cells that expressed an αEβ7 integrin under avariety of buffer conditions. The transfected cells were stained withisotype control antibody (human IgG1) in standard staining buffer(PBS/5% FBS) (FIG. 1A), with mAb 3G6 (IgG1) in standard staining buffer(FIG. 1B) or in buffer that contained EDTA (5 mM; FIG. 1C), in bufferthat contained MnCl₂ (1 mM; FIG. 1D), in buffer that contained MgCl₂ (1mM; FIG. 1E), in buffer that contained CaCl₂ (1 mM; FIG. 1F), in bufferthat contained MgCl₂ and CaCl₂ (1 mM each; FIG. 1G) or in buffer thatcontained MgCl₂, CaCl₂ and MnCl₂ (1 mM each; FIG. 1H), and boundantibody was detected using a fluorescein isothiocyanate (FITC) labeledanti-human IgG antibody. The results show that binding of mAb 3G6 (IgG1)to the transfected cells was enhanced in buffer that contained Mn²⁺(FIGS. 1D and 1H) and inhibited in buffer that contained EDTA (FIG. 1C)relative to binding in standard buffer.

[0016]FIG. 2A is an illustration of a nucleic acid sequence encoding themature heavy chain variable region of mAb 3G6 (SEQ ID NO: 3) and theencoded amino acid sequence of the mature heavy chain variable region ofmAb 3G6 (SEQ ID NO: 4). Complementarity determining region (CDR) 1consists of amino acid residues 31-35 of SEQ ID NO: 4 (SEQ ID NO: 5),CDR 2 consists of amino acid residues 50-66 of SEQ ID NO: 4 (SEQ ID NO:6), CDR 3 consists of amino acid residues 99-112 of SEQ ID NO: 4 (SEQ IDNO: 7).

[0017]FIG. 2B is an illustration of a nucleic acid sequence encoding themature kappa light chain variable region of mAb 3G6 (SEQ ID NO: 8) andthe encoded amino acid sequence of the mature light chain variableregion of mAb 3G6 (SEQ ID NO: 9). Complementarity determining region(CDR) 1 consists of amino acid residues 24-34 of SEQ ID NO: 9 (SEQ IDNO: 10), CDR 2 consists of amino acid residues 50-56 of SEQ ID NO: 9(SEQ ID NO: 11), CDR 3 consists of amino acid residues 89-98 of SEQ IDNO: 9 (SEQ ID NO: 12).

[0018]FIG. 3A is an illustration of a nucleic acid sequence encoding themature heavy chain variable region of mAb 5E4 (SEQ ID NO:13) and theencoded amino acid sequence of the mature heavy chain variable region ofmAb 5E4 (SEQ ID NO: 14). Complementarity determining region (CDR) 1consists of amino acid residues 31-35 of SEQ ID NO: 14 (SEQ ID NO: 15),CDR 2 consists of amino acid residues 50-66 of SEQ ID NO: 14 (SEQ ID NO:16), CDR 3 consists of amino acid residues 99-107 of SEQ ID NO: 14 (SEQID NO: 17).

[0019]FIG. 3B is an illustration of a nucleic acid sequence encoding themature kappa light chain variable region of mAb 5E4 (SEQ ID NO: 18) andthe encoded amino acid sequence of the mature light chain variableregion of mAb 5E4 (SEQ ID NO: 19). Complementarity determining region(CDR) 1 consists of amino acid residues 24-34 of SEQ ID NO: 19 (SEQ IDNO: 20), CDR 2 consists of amino acid residues 50-56 of SEQ ID NO: 19(SEQ ID NO: 21), CDR 3 consists of amino acid residues 89-98 of SEQ IDNO: 19 (SEQ ID NO: 22).

[0020]FIG. 4A is an illustration of a nucleic acid sequence encoding themature heavy chain variable region of mAb 8D5 (SEQ ID NO: 23) and theencoded amino acid sequence of the mature heavy chain variable region ofmAb 8D5 (SEQ ID NO: 24). Complementarity determining region (CDR) 1consists of amino acid residues 31-35 of SEQ ID NO: 24 (SEQ ID NO: 25),CDR 2 consists of amino acid residues 50-65 of SEQ ID NO: 24 (SEQ ID NO:26), CDR 3 consists of amino acid residues 98-117 of SEQ ID NO: 24 (SEQID NO: 27).

[0021]FIG. 4B is an illustration of a nucleic acid sequence encoding themature kappa light chain variable region of mAb 8D5 (SEQ ID NO: 28) andthe encoded amino acid sequence of the mature light chain variableregion of mAb 8D5 (SEQ ID NO: 29). Complementarity determining region(CDR) 1 consists of amino acid residues 24-34 of SEQ ID NO: 29 (SEQ IDNO: 30), CDR 2 consists of amino acid residues 50-56 of SEQ ID NO: 29(SEQ ID NO: 31), CDR 3 consists of amino acid residues 89-97 of SEQ IDNO: 29 (SEQ ID NO: 32).

DETAILED DESCRIPTION OF THE INVENTION

[0022] As used herein, “activation-induced epitope” refers to an epitopethat is present on an activated αE integrin (e.g., integrin αE chain(CD103), an αEβ7 integrin) but not on non-activated αE integrin. Anactivated αE integrin is an αE integrin that binds ligand (e.g.,E-cadherin) with high affinity, while a non-activated αE integrin bindsthe ligand with low affinity. (See, Higgins, J. M. G. et al., J. Biol.Chem. 140:197-210 (1998).) An αE integrin can be activated, for example,by exposure to divalent cations (e.g., Mn²⁺). When the αE integrin isexpressed on the surface of a cell, it can be activated upon exposure ofthe cell to phorbol esters (e.g., Phorbol 12-myristate 13-acetate(PMA)), or to suitable growth factors and/or mitogens (e.g.,concanavalin A). An αE integrin expressed on a T cell can be activatedby signals transduced through the T cell receptor (TCR) complex (e.g.,upon TCR binding to specific MHC-peptide complexes, crosslinking withanti-CD3 antibody).

[0023] An antibody that “binds an activation-induced epitope” onintegrin αE chain binds integrin αE chain under activation conditions(e.g., in the presence of divalent cations (e.g., Mn²⁺)) but does notsignificantly bind in the absence of activation (e.g., when a suitablechelating agent (e.g., Ethylenediaminetetraacetic acid (EDTA)) ispresent).

[0024] As used herein, an antibody and antigen-binding fragment thereofthat “binds” an αE integrin (e.g., an activated αE integrin, an αEβ7integrin, an integrin αE chain (CD103)) has binding specificity for theαE integrin. The terms “binding specificity” or “specific” whenreferring to an antibody-antigen interaction indicate that the antibodycan discriminate between one or more αE integrins (e.g., an activated αEintegrin, an αEβ7 integrin, an integrin αE chain (CD103)) and otherantigens, rather than to indicate that the antibody can bind only oneantigen. For example, in certain embodiments, the antibody orantigen-binding fragments of the invention can “selectively bind” an αEintegrin. Such selective antibodies or antigen-binding fragments maybind another antigen with low affinity, but bind said αE integrin withhigher affinity. Under appropriate binding conditions (e.g.,physiological conditions), an antibody or antigen-binding fragmentthereof that selectively binds an αE integrin will bind the αE integrinbut will not significantly bind other antigens. An antibody orantigen-binding fragment of an antibody does not “significantly bind” anantigen when the extent of binding is less than about 25%, preferablyless than about 15%, more preferably less than about 10%, mostpreferably less than about 5% or less than about 2% or 1% of the levelof binding to an antigen that is “selectively” bound under the sameconditions (e.g., physiological conditions). The concentration ofantibody and other conditions required to provide selectivity for an αEintegrin (e.g., an antibody concentration and pH which reduces oreliminates non-selective binding) can be readily determined using anysuitable method, such as titration.

[0025] As used herein, the term “functionally rearranged” refers to asegment of DNA from an immunoglobulin locus which has undergone V(D)Jrecombination, with or without insertion or deletion of nucleotide(s)(e.g., N nucleotides, P nucleotides) and/or somatic mutation, therebyproducing an immunoglobulin gene which encodes an immunoglobulinvariable region or immunoglobulin chain (e.g., heavy chain, lightchain). A functionally rearranged immunoglobulin gene can be directly orindirectly identified using suitable methods, such as, for example,nucleotide sequencing, hybridization (e.g., Southern blotting, Northernblotting) using probes which can anneal to coding joints between genesegments (e.g., VH, VL, D, JH, JL) or enzymatic amplification ofimmunoglobulin genes (e.g., polymerase chain reaction) with primerswhich can anneal to coding joints between gene segments. Whether a cellproduces an antibody comprising a particular variable region or avariable region comprising a particular sequence (e.g., a CDR sequence)can also be determined using suitable methods. In one example, mRNA canbe isolated from an antibody producing cell (e.g., a hybridoma) and usedto produce cDNA. The cDNA can be cloned and sequenced or can beamplified (e.g., by polymerase chain reaction) using a first primerwhich anneals specifically to a portion of the variable region ofinterest (e.g., CDR, coding joint) and a second primer which annealsspecifically to non-variable region sequences (e.g., C_(H)1, C_(L)).

[0026] As used herein, the phrase “of human origin” refers toantibodies, antigen-binding fragments of antibodies and portions orregions of antibodies (e.g., variable regions, complementaritydetermining regions (CDRs), framework regions (FRs), constant regions)having amino acid sequences that are encoded by nucleotide sequencesderived from human (Homo sapiens) germ line immunoglobulin genes. Forexample, an antibody of human origin can be encoded by human germ lineimmunoglobulin genes that have been functionally rearranged to produce afunctional gene that can be expressed to produce an antibody. Asdescribed herein, functionally rearranged genes that encode an antibodychain can include sequences that are not found in the germ line, such asN nucleotides and P nucleotides, and mutations that can occur as part ofthe processes that produce high-affinity antibodies (e.g., somaticmutation, affinity maturation, clonal selection). Functionallyrearranged immunoglobulin genes of human origin, including those thatinclude non-germ line sequences, can be generated via natural processesin a suitable in vivo expression system (e.g., a human, a human-antibodytransgenic animal), artificially using any suitable methods (e.g.,recombinant DNA technology, phage display) or any combination of naturaland artificial processes. Antibodies, antigen-binding fragments ofantibodies and portions or regions of antibodies of human origin can beproduced, for example, by expression of a nucleic acid of non-humanorigin (e.g., a synthetic nucleic acid) that has the requisitenucleotide sequence.

[0027] An antibody, antigen-binding fragment of an antibody or a portionof an antibody (e.g., a framework region) “of human origin” can have anamino acid sequence that is encoded by a nucleic acid that has anucleotide sequence that is a consensus of the nucleotide sequences of anumber of naturally occurring human antibody genes or human germ linesequences, or have an amino acid sequence that is a consensus of theamino acid sequences of a number of naturally occurring human antibodiesor amino acid sequences encoded in the human germ line. A number ofhuman antibody consensus sequences are available, including consensussequences for the different subgroups of human variable regions (see,Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, U.S.Government Printing Office (1991). The Kabat database and itsapplications are freely available on line. (See, Johnson, G. and Wu, T.T., Nucleic Acids Research 29:205-206 (2001).)

[0028] As used herein, the phrase “human antibody” refers to antibodiesor antigen-binding fragments of antibodies in which the variable andconstant regions (if present) have amino acid sequences that are encodedby nucleotide sequences derived from human (Homo sapiens) germlineimmunoglobulin genes. A “human antibody” can include sequences that arenot encoded in the germline (e.g., due to N nucleotides, P nucleotides,and mutations that can occur as part of the processes that producehigh-affinity antibodies such as, somatic mutation, affinity maturation,clonal selection)) that occur as a result of biological processes in asuitable in vivo expression system (e.g., a human, a human-antibodytransgenic animal). Antibodies, antigen-binding fragments of antibodiesand portions or regions of human antibodies can be produced, forexample, by expression of a nucleic acid of non-human origin (e.g., asynthetic nucleic acid) that has the requisite nucleotide sequence.

[0029] As used herein, the phrase “CDR-grafted” antibody refers toantibodies and antigen-binding fragments of antibodies that comprise aCDR that is not naturally associated with the framework regions of theantibody or antigen-binding fragment. Generally the CDR is from anantibody from a first species and the framework regions and constantregions (if present) are from an antibody from a different species. TheCDR-grafted antibody can be a “humanized antibody.”

[0030] As used herein, “humanized antibody” refers to an antibody orantigen-binding fragment thereof comprising a CDR that is not of humanorigin and framework and/or constant regions that are of human origin.For example, a humanized antibody can comprise a CDR derived from anantibody of nonhuman origin (e.g., natural antibody such as a murine(e.g., mouse, rat) antibody, artificial antibody) that binds an αEintegrin, preferably integrin αE chain (CD103), and framework andconstant regions (if present) of human origin (e.g., a human frameworkregion, a human consensus framework region, a human constant region(e.g., CL, CH1, hinge, CH2, CH3, CH4)). CDR-grafted single chainantibodies containing a CDR of non-human origin and framework andconstant regions (if present) of human origin (e.g., CDR-grafted scFV)are also encompassed by the term humanized antibody.

[0031] As used herein, the term “chimeric antibody” refers to anantibody or antigen-binding fragment thereof comprising a variableregion from an antibody from a first species and a constant region froman antibody from a different species. None of the portions whichcomprise a chimeric antibody need to be of human origin. For example, achimeric antibody can comprise a variable region from a rodent (e.g.,mouse) antibody and a constant region of a non-human primate antibody(e.g., a chimpanzee constant region).

[0032] The antibody of the invention can be a single chain antibody(e.g., a single chain Fv (scFv)) and can include a linker moiety (e.g.,a linker peptide) not found in native antibodies. For example, an scFvcan comprise a linker peptide, such as two to about twenty glycineresidues or other suitable linker, which connects a heavy chain variableregion to a light chain variable region. For the purposes of theinvention, the presence of such a linker does not affect the status ofthe single chain antibody as being “of human origin” or “human.” Forexample, a human scFv can comprise a human heavy chain variable regionand a human light chain variable region that are connected through asuitable peptide linker.

[0033] “Conservative amino acid substitution” refers to the replacementof a first amino acid by a second amino acid that has chemical and/orphysical properties (e.g., charge, structure, polarity,hydrophobicity/hydrophilicity) which are similar to those of the firstamino acid. For example, replacement of one amino acid by another withinthe following groups is a conservative amino acid substitution: Ala,Val, Leu, and Ile; Ser and Thr; Asp and Glu; Asn and Gln; Lys and Arg;Phe and Tyr.

[0034] A nucleotide sequence encoding a human (Homo sapiens) integrin αEchain (CD103), used in the studies described herein and deposited inGenBank under accession number L25851, is presented as SEQ ID NO: 1.(See also, Shaw et al., J. Biol. Chem. 269:6016-6025 (1994).) Thenucleotide sequence has an open-reading frame beginning at position 126.The amino acid sequence of a human integrin αE chain encoded by SEQ IDNO: 1 is presented as SEQ ID NO: 2. The human integrin αE chain containsa signal peptide (amino acid residues −18 to −1 of SEQ ID NO: 2), anX-domain (amino acid residues 126-180 of SEQ ID NO: 2) and an I-Domain(residues 181-372 of SEQ ID NO: 2). The entire teachings of GenBankAccession No. L25851 are incorporated herein by reference.

[0035] A nucleotide sequence encoding a human (Homo sapiens) E-cadherinused in the studies described herein and deposited in GenBank underaccession number L08599 is presented as SEQ ID NO:33. The nucleotidesequence has an open-reading frame beginning at position 109. The aminoacid sequence of a human E-cadherin encoded by SEQ ID NO: 33 ispresented as SEQ ID NO: 34. The entire teachings of GenBank AccessionNo. L08599 are incorporated herein by reference.

[0036] A nucleotide sequence encoding a human (Homo sapiens) integrin α4chain used in the studies described herein and deposited in GenBankunder accession number L12002 is presented as SEQ ID NO: 35. Thenucleotide sequence has an open-reading frame beginning at position 411.The amino acid sequence of an integrin α4 chain encoded by SEQ ID NO: 35is presented as SEQ ID NO:36. The entire teachings of GenBank AccessionNo. L12002 are incorporated herein by reference.

[0037] A nucleotide sequence encoding a human (Homo sapiens) integrin β7chain used in the studies described herein and deposited in GenBankunder accession number M62880 is presented as SEQ ID NO:37. Thenucleotide sequence has an open-reading frame beginning at position 114.The amino acid sequence of an integrin β7 chain encoded by SEQ ID NO: 37is presented as SEQ ID NO: 38. The entire teachings of GenBank AccessionNo. M62880 are incorporated herein by reference.

[0038] Antibodies and Antibody Producing Cells

[0039] The antibody of the invention can be polyclonal or monoclonal,and the term “antibody” is intended to encompass both polyclonal andmonoclonal antibodies. The terms polyclonal and monoclonal refer to thedegree of homogeneity of an antibody preparation, and are not intendedto be limited to particular methods of production. The term “antibody”as used herein encompasses antigen-binding fragments of antibodies,including antigen-binding fragments of human, humanized, chimeric,CDR-grafted, veneered or single-chain antibodies.

[0040] Antibodies which bind an αE integrin can be selected from asuitable collection of natural or artificial antibodies or raisedagainst an appropriate immunogen in a suitable host. For example,antibodies can be raised by immunizing a suitable host (e.g., mouse,human antibody-transgenic mouse) with a suitable immunogen, such as anisolated or purified αE integrin (e.g., αEβ7) or cells expressing arecombinant αE integrin (e.g., cell that expresses an exogenous nucleicacid encoding human integrin αE chain (CD103)). In addition, cellsexpressing a recombinant αE integrin, such as transfected cells, can beused in a screen for antibody which binds thereto (See e.g.,Chuntharapai et al., J. Immunol., 152: 1783-1789 (1994); Chuntharapai etal., U.S. Pat. No. 5,440,021).

[0041] Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described. (See, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976);Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat.No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991).) Generally, where a monoclonal antibody is desired,a hybridoma is produced by fusing a suitable immortal cell line (e.g., amyeloma cell line such as SP2/0, P3X63Ag8.653 or a heteromyeloma) withantibody-producing cells. Antibody-producing cells can be obtained fromthe peripheral blood or, preferably the spleen or lymph nodes, ofhumans, human-antibody transgenic animals or other suitable animalsimmunized with the antigen of interest. Cells that produce antibodies ofhuman origin (e.g., a human antibody) can be produced using suitablemethods, for example, fusion of a human antibody-producing cell and aheteromyeloma or trioma, or immortalization of an activated human B cellvia infection with Epstein Barr virus. (See, e.g., U.S. Pat. No.6,197,582 (Trakht); Niedbala et al., Hybridoma, 17:299-304 (1998);Zanella et al., J Immunol Methods, 156:205-215 (1992); Gustafsson etal., Hum Antibodies Hybridomas, 2:26-32 (1991).) The fused orimmortalized antibody-producing cells (hybridomas) can be isolated usingselective culture conditions, and cloned by limiting dilution. Cellswhich produce antibodies with the desired specificity can be identifiedusing a suitable assay (e.g., ELISA).

[0042] Other suitable methods of producing or isolating antibodies orantigen-binding fragments of the desired specificity can be used,including, for example, methods which select a recombinant antibody orantigen-binding fragment thereof from a library, such as a phage displaylibrary. Such libraries can contain antibodies or antigen-bindingfragments of antibodies that contain natural or artificial amino acidsequences. For example, the library can contain Fab fragments whichcontain artificial CDRs (e.g., random amino acid sequences) and humanframework regions. (See, for example, U.S. Pat. No. 6,300,064 (Knappik,et al.), the entire teachings of which are incorporated herein byreference.)

[0043] Human antibodies and nucleic acids encoding same can be obtainedfrom a human or from human-antibody transgenic animals. Human-antibodytransgenic animals (e.g., mice) are animals that are capable ofproducing a repertoire of human antibodies, such as XENOMOUSE (Abgenix,Fremont, Calif.), HUMAB-MOUSE, KIRIN TC MOUSE or KM-MOUSE (MEDAREX,Princeton, N.J.). Generally, the genome of human-antibody transgenicanimals has been altered to include a transgene comprising DNA from ahuman immunoglobulin locus that can undergo functional rearrangement. Anendogenous immunoglobulin locus in a human-antibody transgenic animalcan be disrupted or deleted to eliminate the capacity of the animal toproduce antibodies encoded by an endogenous gene. Suitable methods forproducing human-antibody transgenic animals are well known in the art.(See, for example, U.S. Pat. Nos. 5,939,598 and 6,075,181 (Kucherlapatiet al.), U.S. Pat. Nos. 5,569,825, 5,545,806, 5,625,126, 5,633,425,5,661,016, and 5,789,650 (Lonberg et al.), Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555 (1993), Jakobovits et al., Nature,362: 255-258 (1993), Jakobovits et al. WO 98/50433, Jakobovits et al. WO98/24893, Lonberg et al. WO 98/24884, Lonberg et al. WO 97/13852,Lonberg et al. WO 94/25585, Lonberg et al. EP 0 814 259 A2, Lonberg etal. GB 2 272 440 A, Lonberg et al., Nature 368:856-859 (1994), Lonberget al., Int Rev Immunol 13(1):65-93 (1995), Kucherlapati et al. WO96/34096, Kucherlapati et al. EP 0 463 151 B1, Kucherlapati et al. EP 0710 719 A1, Surani et al. U.S. Pat. No. 5,545,807, Bruggemann et al. WO90/04036, Bruggemann et al. EP 0 438 474 B1, Taylor et al., Int.Immunol. 6(4)579-591 (1994), Taylor et al., Nucleic Acids Research20(23):6287-6295 (1992), Green et al, Nature Genetics 7:13-21 (1994),Mendez et al., Nature Genetics 15:146-156 (1997), Tuaillon et al., ProcNatl Acad Sci USA 90(8)3720-3724 (1993) and Fishwild et al., NatBiotechnol 14(7):845-851 (1996), the teachings of each of the foregoingare incorporated herein by reference in their entirety.)

[0044] As described herein, human-antibody transgenic animals can beimmunized with a suitable composition comprising an antigen of interest(e.g., a recombinant cell expressing an αEβ7 integrin). Antibodyproducing cells can be isolated and fused to form hybridomas usingconventional methods. Hybridomas that produce human antibodies havingthe desired characteristics (e.g., specificity, affinity) can beidentified using any suitable assay (e.g, ELISA) and, if desired,selected and subcloned using suitable culture techniques.

[0045] Human-antibody transgenic animals provide a source of nucleicacids that can be enriched in nucleic acids that encode antibodieshaving desired properties, such as specificity and affinity. Forexample, nucleic acids encoding antibodies or antibody variable regionscan be isolated from human-antibody transgenic mice that have beenimmunized with an αE integrin. The isolated nucleic acids or portionsthereof (e.g., portions encoding variable regions, CDRs, frameworkregions) can be expressed using any suitable method (e.g., phagedisplay) to produce a library of antibodies or antigen-binding fragmentsof antibodies (e.g., single chain antigen-binding fragments, doublechain antigen-binding fragments) that is enriched for antibodies orantigen-binding fragments that bind αE. Such a library can exhibitenhanced diversity (e.g., combinatorial diversity through pairing ofheavy chain variable regions and light chain variable regions) relativeto the repertoire of antibodies produced in the immunized human-antibodytransgenic animal. The library can be screened using any suitable assay(e.g., an αE binding assay) to identify antibodies or antigen-bindingfragments having desired properties (e.g., specificity, affinity). Thenucleic acids encoding antibody or antigen-binding fragments havingdesired properties can be recovered using any suitable methods. (See,e.g., U.S. Pat. No. 5,871,907 (Winter et al.) and U.S. Pat. No.6,057,098 (Buechler et al.), the entire teachings of each of theforegoing are incorporated herein by reference.)

[0046] The antibody of the invention can be a CDR-grafted (e.g.,humanized) antibody or an antigen-binding fragment thereof. The CDRs ofa CDR-grafted antibody can be derived from a suitable antibody whichbinds an αE integrin (referred to as a donor antibody). For example,suitable CDRs can be derived from mAb 3G6, mAb 5E4 or mAb 8D5 which, asdescribed herein, bind integrin αE chain (CD103) or from any othersuitable antibody. Other sources of suitable CDRs include natural andartificial αE integrin-specific antibodies obtained from nonhumansources, such as rodent (e.g., mouse, rat), rabbit, pig, goat, non-humanprimate (e.g., monkey) or non-human library.

[0047] The framework regions of a CDR-grafted antibody are preferably ofhuman origin, and can be derived from any human antibody variable regionhaving sequence similarity to the analogous or equivalent region (e.g.,light chain variable region) of the antigen binding region of the donorantibody. Other sources of framework regions of human origin includehuman variable region consensus sequences. (See, e.g., Kettleborough, C.A. et al., Protein Engineering 4:773-783 (1991); Carter et al., WO94/04679; Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,U.S. Government Printing Office (1991)).

[0048] In one embodiment, the framework regions of a CDR-grafted (e.g.,humanized) antibody chain can be derived from a variable region of humanorigin having at least about 65% overall amino acid sequence identity,and preferably at least about 70% overall amino acid sequence identity,with the amino acid sequence of the variable region of the donorantibody. A suitable framework region can also be derived from aantibody of human origin having at least about 65% amino acid sequenceidentity, and preferably at least about 70%, 80%, 90% or 95% amino acidsequence identity over the length of the framework region within theamino acid sequence of the equivalent portion (e.g., framework region)of the donor antibody. For example, a suitable framework region of humanorigin can be derived from an antibody of human origin (e.g., a humanantibody) having at least about 65% amino acid sequence identity, andpreferably at least about 70%, 80%, 90% or 95% amino acid sequenceidentity, over the length of the particular framework region being used,when compared to the amino acid sequence of the equivalent portion(e.g., framework region) of the donor antibody. Amino acid sequenceidentity can be determined using a suitable amino acid sequence alignentalgorithm, such as CLUSTAL W, using the default parameters. (Thompson J.D. et al., Nucleic Acids Res. 22:4673-4680 (1994).)

[0049] Framework regions of human origin can include amino acidsubstitutions or replacements, such as “back mutations” which replace anamino acid residue in the framework region of human origin with aresidue from the corresponding position of the donor antibody. One ormore mutations in the framework region can be made, including deletions,insertions and substitutions of one or more amino acids. Preferably, theCDR-grafted (e.g., humanized) antibody binds αE integrin with anaffinity similar to, substantially the same as, or better than that ofthe donor antibody. Variants can be produced by a variety of suitablemethods, including mutagenesis of nonhuman donor or acceptor humanchains. (See, e.g., U.S. Pat. Nos. 5,693,762 (Queen et al.) and5,859,205 (Adair et al.), the entire teachings of which are incorporatedherein by reference.)

[0050] Constant regions of antibodies, antibody chains (e.g, heavychain, light chain) or fragments or portions thereof of the invention,if present, can be derived from any suitable source. For example,constant regions of human, humanized and certain chimeric antibodies,antibody chains (e.g, heavy chain, light chain) or fragments or portionsthereof, if present can be of human origin and can be derived from anysuitable human antibody or antibody chain. For example, a constantregion of human origin or portion thereof can be derived from a human κor λ light chain, and/or a human γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g.,α1, α2), δ or ε heavy chain, including allelic variants. In certainembodiments, the antibody or antigen-binding fragment (e.g., antibody ofhuman origin, human antibody) can include amino acid substitutions orreplacements that alter or tailor function (e.g., effector function).For example, a constant region of human origin (e.g., γ1 constantregion, γ2 constant region) can be designed to reduce complementactivation and/or Fc receptor binding. (See, for example, U.S. Pat. Nos.5,648,260 (Winter et al.), 5,624,821 (Winter et al.) and 5,834,597 (Tsoet al.), the entire teachings of which are incorporated herein byreference.) Preferably, the amino acid sequence of a constant region ofhuman origin that contains such amino acid substitutions or replacementsis at least about 95% identical over the full length to the amino acidsequence of the unaltered constant region of human origin, morepreferably at least about 99% identical over the full length to theamino acid sequence of the unaltered constant region of human origin.

[0051] Humanized antibodies or antigen-binding fragments of a humanizedantibody can be prepared using any suitable method. Several such methodsare well-known in the art. (See, e.g., U.S. Pat. No. 5,225,539 (Winter),U.S. Pat. No. 5,530,101 (Queen et al.).) The portions of a humanizedantibody (e.g., CDRs, framework, constant region) can be obtained orderived directly from suitable antibodies (e.g., by de novo synthesis ofa portion), or nucleic acids encoding an antibody or chain thereofhaving the desired property (e.g., binds αE integrin) can be producedand expressed. Humanized immunoglobulins comprising the desired portions(e.g., CDR, FR, constant region) of human and nonhuman origin can beproduced using synthetic and/or recombinant nucleic acids to prepare anucleic acid (e.g., cDNA) encoding the desired humanized chain. Toprepare a portion of a chain, one or more stop codons can be introducedat the desired position. For example, nucleic acid (e.g., DNA) sequencescoding for newly designed humanized variable regions can be constructedusing PCR mutagenesis methods to alter existing DNA sequences. (See,e.g., Kamman, M., et al., Nucl. Acids Res. 17:5404 (1989).) PCR primerscoding for the new CDRs can be hybridized to a DNA template of apreviously humanized variable region which is based on the same, or avery similar, human variable region (Sato, K., et al., Cancer Research53:851-856 (1993)). If a similar DNA sequence is not available for useas a template, a nucleic acid comprising a sequence encoding a variableregion sequence can be constructed from synthetic oligonucleotides (seee.g., Kolbinger, F., Protein Engineering 8:971-980 (1993)). A sequenceencoding a signal peptide can also be incorporated into the nucleic acid(e.g., on synthesis, upon insertion into a vector). The natural signalpeptide sequence from the acceptor antibody, a signal peptide sequencefrom another antibody or other suitable sequence can be used (see, e.g.,Kettleborough, C. A., Protein Engineering 4:773-783 (1991)). Using thesemethods, methods described herein or other suitable methods, variantscan be readily produced. In one embodiment, cloned variable regions canbe mutated, and sequences encoding variants with the desired specificitycan be selected (e.g., from a phage library; see, e.g., U.S. Pat. No.5,514,548 (Krebber et al.) and WO 93/06213 (Hoogenboom et al.)).

[0052] The antibody of the invention can be a chimeric antibody or anantigen-binding fragment of a chimeric antibody. Preferably, thechimeric antibody or antigen-binding fragment thereof comprises avariable region of non-human origin and a constant region of humanorigin (e.g., a human constant region).

[0053] Chimeric antibodies and antigen-binding fragments of chimericantibodies that bind αE integrin can be prepared using any suitablemethod. Several suitable methods are well-known in the art. (See, e.g.,U.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,116,946 (Caponet al.).) Generally, chimeric antibodies are produced by preparing, foreach of the light and heavy chain components of the chimericimmunoglobulin, a recombinant nucleic acid comprising a first nucleotidesequence encoding at least the variable region of an antibody from afirst species that binds αE integrin that is joined in frame to a secondnucleotide sequence encoding at least a part of a constant region froman antibody of a different species. Generally, the recombinant nucleicacid encodes a chimeric heavy chain or a chimeric light chain. However,if desired, a single recombinant nucleic acid encoding a chimeric heavychain and a chimeric light chain can be prepared. The recombinantnucleic acids can be assembled in or inserted into an expression vector.The recombinant nucleic acid(s) can be introduced into a suitable hostcell that is capable of expressing the chimeric antibody or chimericantibody chain using any suitable method (e.g., transfection,transformation, infection) to produce a recombinant host cell. Therecombinant host cell can be maintained under conditions suitable forexpression of the chimeric antibody or chimeric antibody chain and theantibody or chain can be recovered.

[0054] Nucleic acids encoding the variable region of antibody light andheavy chains can be obtained from cells (e.g., B cells, hybridoma cells)that produce an antibody that binds αE integrin. For example, nucleicacids that encode human heavy and light chain variable regions that canbind αE integrin can be obtained from hybridomas 3G6, 5E4 and 8D5, andfrom recombinant cell lines CHO 3G6 C1.2D6 and CHO 5E4 A1.2C12,described herein. Nucleic acids that encode constant regions can beobtained from suitable sources using any suitable technique, such aconventional techniques of recombinant DNA technology. The nucleotidesequences of nucleic acids encoding human κ or λ light chain constantregions, and γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g., α1, α2), δ or ε humanheavy chain constant regions are readily available.

[0055] The invention also relates to a bispecific antibody orantigen-binding fragment thereof (e.g., F(ab′)₂), which binds an αEintegrin and at least one other antigen. In a particular embodiment, thebispecific antibody, or antigen-binding fragment thereof binds anactivation-induced epitope on an αE integrin (e.g., integrin αE chain(CD103)). In other embodiments, the bispecific antibody orantigen-binding fragment thereof has the epitopic specificity of mAb3G6, mAb 5E4 or mAb 8D5 and at least one other antibody. Bispecificantibodies can be secreted by triomas and hybrid hybridomas. Generally,triomas are formed by fusion of a hybridoma and a lymphocyte (e.g.,antibody secreting B cell) and hybrid hybridomas are formed by fusion oftwo hybridomas. Each of the cells that are fused to produce a trioma orhybrid hybridoma produces a monospecific antibody. However, triomas andhybrid hybridomas can produce an antibody containing antigen bindingsites which recognize different antigens. The supernatants of triomasand hybrid hybridomas can be assayed for bispecific antibody using asuitable assay (e.g., ELISA), and bispecific antibodies can be purifiedusing conventional methods. (See, e.g., U.S. Pat. No. 5,959,084 (Ring etal.) U.S. Pat. No. 5,141,736 (Iwasa et al.), U.S. Pat. Nos. 4,444,878,5,292,668 and 5,523,210 (Paulus et al.) and U.S. Pat. No. 5,496,549(Yamazaki et al.).)

[0056] The various portions of an antibody (e.g., mouse antibody, humanantibody, humanized antibody, chimeric antibody and antigen-bindingfragments of the foregoing) can be joined together chemically usingconventional techniques, or can be prepared as a continuous polypeptidechain by expression (in vivo or in vitro) of a nucleic acid (one or morenucleic acids) encoding antibody. For example, nucleic acids encoding ahuman, humanized or chimeric chain can be expressed in vivo or in vitroto produce a continuous polypeptide chain. See, e.g., Cabilly et al.,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European PatentNo. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S.et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No.5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al.,European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596A1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460 (1992),regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988))regarding single chain antibodies.

[0057] The invention also relates to antigen-binding fragments ofantibodies that retain the capacity to bind antigen (e.g., an αEintegrin, an activation-induced epitope on integrin αE chain). Suchantigen-binding fragments of antibodies retain the antigen bindingfunction of a corresponding full-length antibody (e.g., bindingspecificity for an αE integrin), and preferably inhibit binding ofligand (e.g., E-cadherin) to an αE integrin (e.g., αEβ7).Antigen-binding fragments of antibodies encompassed by the inventioninclude, Fv fragments (e.g., single chain Fv fragments (scFv)), Fabfragments, Fab′ fragments and F(ab′)₂ fragments, for example. Suchantigen-binding fragments can be produced using any suitable method, forexample by enzymatic cleavage and/or using recombinant DNA technology.For example, an antibody can be cleaved with papain or pepsin to yield aFab fragment or F(ab′)₂ fragment, respectively. Other proteases with therequisite substrate specificity can also be used to generateantigen-binding fragments of antibodies, such as Fab fragments orF(ab′)₂ fragments. Similarly, Fv fragments can be prepared by digestingan antibody with a suitable protease or using recombinant DNAtechnology. For example, a nucleic acid can be prepared that encodes alight chain variable region and heavy chain variable region that areconnected by a suitable peptide linker, such as a chain of two to abouttwenty Glycyl residues. The nucleic acid can be introduced into asuitable host (e.g., E. coli) using any suitable technique (e.g.,transfection, transformation, infection), and the host can be maintainedunder conditions suitable for expression of a single chain Fv fragment.A variety of antigen-binding fragments of antibodies can be preparedusing antibody genes in which one or more stop codons has beenintroduced upstream of the natural stop site. For example, an expressionconstruct encoding a F(ab′)₂ portion of an immunoglobulin heavy chaincan be designed by introducing a translation stop codon at the 3′ end ofthe sequence encoding the hinge region of the heavy chain.

[0058] The invention also relates to the individual heavy and lightchains of the antibodies (e.g., mouse antibodies, human antibodies,humanized antibodies, chimeric antibodies) that bind an αE integrin andto antigen-binding portions thereof. The heavy chains or light chains(and antigen-binding portions thereof) of the invention can bind an αEintegrin when paired with a complementary light or heavy chain,respectively. Complementary chains can be identified using any suitablemethod (e.g., phage display, transgenic animals). For example, atransgenic animal comprising a functionally rearranged nucleic acidencoding a desired heavy chain can be prepared. The heavy-chaintransgenic animal can be immunized with the antigen of interest andhybridomas produced. Because of allelic exclusion at immunoglubulinloci, the heavy-chain transgenic mouse may not significantly expressendogenous heavy chains and substantially all antibodies elicited byimmunization can comprise the heavy chain of interest and acomplementary light chain.

[0059] The antigen-binding properties (e.g., specificity, affinity) ofantibodies and antigen-binding fragments of antibodies can be elucidatedusing any suitable method. For example, binding specificity can bedetermined using assays in which formation of a complex between antibodyor antigen-binding fragment and an αE integrin, such as an αEβ7integrin, is detected or measured. Compositions which comprise an αEintegrin and which can be used to assess antigen-binding properties ofthe antibodies and antigen-binding fragments described herein include, amembrane fraction of a cell comprising an αEβ7 integrin, a cell bearingan αEβ7 integrin, such as a human lymphocyte, human lymphocyte cell lineor recombinant host cell comprising a nucleic acid encoding αE and/or β7which expresses an αEβ7 integrin, a recombinant soluble αEβ7, such asts.αEβ7.coil described herein, and the like. Binding and/or adhesionassays or other suitable methods can also be used in procedures for theidentification and/or isolation of antibodies (e.g., human and/orhumanized antibodies) having the requisite specificity (e.g., an assayin which adhesion between a cell bearing an αEβ7 integrin and a ligandthereof (e.g., a second cell expressing E-cadherin, an immobilizedE-cadherin fusion protein (e.g., E-cadherin-Fc fusion protein) isdetected and/or measured), or other suitable methods.

[0060] The antibodies of the invention bind an αE integrin (e.g., αEβ7)and preferably bind integrin αE chain (CD103). In a preferredembodiment, the antibody or antigen-binding fragment selectively bindsan activation-induced epitope on an integrin αE chain (CD103). Theactivation-induced epitope can be induced by activation with a divalentcation, such as Mn²⁺, Mg⁺, Ca²⁺ or any combination of the foregoing. Theactivation-induced epitope on an integrin αE chain expressed on thesurface of a cell (e.g., as integrin αEβ7) can also be induced byexposing the cell to phorbol esters (e.g., PMA), or suitable mitogensand/or growth factors. When the cell expressing an integrin αE chain isa T cell, the activation-induced epitope can be induced by signalstransduced through the T cell receptor complex. Thus, antibodies thatselectively bind an activation-induced epitope can be used to detect oridentify activated T cells that express an αE integrin for diagnosticand/or therapeutic purposes.

[0061] In one embodiment, the antibody of the invention binds anactivation-induced epitope that is induced by exposure of the αEintegrin to a divalent cation. Such antibodies bind an integrin αE chain(CD103) in the presence of a divalent cation, such as Mn²⁺, but do notsignificantly bind an integrin αE chain in the absence of a divalentcation or in the presence of a suitable divalent cation chelating agent(e.g., EDTA).

[0062] In certain embodiments, the antibody selectively binds anactivation-induced epitope on an integrin αE chain that comprises aminoacid residues in the I domain (amino acids 199-390 of SEQ ID NO:2) ofintegrin αE chain.

[0063] In other embodiments, the antibody binds an αE integrin (e.g.,selectively binds an activation-induced epitope on integrin αE chain)and inhibits binding of ligand, such as E-cadherin, to the αE integrin(e.g., αEβ7 integrin). For example, the antibody can inhibit αE integrinmediated adhesion of a cell expressing an αE integrin (e.g., αEβ7) tocells expressing a ligand for an αE integrin (e.g. E-cadherin), such asepithelial cells and/or endothelial cells. Preferably, the antibodies donot bind the X domain of integrin αE chain (amino acids 144-198 of SEQID NO: 2).

[0064] Preferred antibodies that bind an αE integrin (e.g., selectivelybind an activation-induced epitope on an integrin αE chain) includechimeric antibodies, humanized antibodies and antigen-binding fragmentsof the foregoing. Particularly preferred antibodies are human antibodiesand antigen-binding fragments of human antibodies.

[0065] As described herein, human antibodies designated mAb 3G6, mAb 5E4and mAb 8D5 which bind integrin αE chain (CD103) have been produced. mAb3G6 and mAb 5E4 were originally produced as IgM antibodies and mAb 8D5was originally produced as an IgG2 antibody. As described herein, IgG1forms of mAbs 3G6, 5E4 and 8D5 have also been produced.

[0066] mAb 3G6 (IgM) can be produced by hybridoma 3G6, also referred toas hybridoma 241 3G6.1.15, which was deposited on Apr. 3, 2002, onbehalf of Millennium Pharmaceuticals Inc., 75 Sidney Street, Cambridge,Mass., 02139, USA, at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110, U.S.A., under Accession No.PTA-4201. The invention relates to hybridoma 3G6, to the antibody itproduces, antigen-binding fragments thereof, and to nucleic acidsencoding the antibody and portions thereof (e.g., heavy chain, heavychain variable region, light chain, light chain variable region).

[0067] An IgG1 form of mAb 3G6 can be produced by “3G6 CHO stable cellline,” also referred to as CHO 3G6 C1.2D6, which was deposited on Apr.3, 2002, on behalf of Millennium Pharmaceuticals Inc., 75 Sidney Street,Cambridge, Mass., 02139, USA, at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110, U.S.A., under AccessionNo. PTA-4204. The invention relates to cell line CHO 3G6 C1.2D6, to theantibody it produces, antigen-binding fragments thereof, and to nucleicacids encoding the antibody and portions thereof (e.g., heavy chain,heavy chain variable region, light chain, light chain variable region).

[0068] mAb 5E4 can be produced by hybridoma 5E4, also referred to ashybridoma 233 5E4.3.10, which was deposited on Apr. 3, 2002, on behalfof Millennium Pharmaceuticals Inc., 75 Sidney Street, Cambridge, Mass.,02139, USA, at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110, U.S.A., under Accession No. PTA-4202.The invention relates to hybridoma 5E4, to the antibody it produces,antigen-binding fragments thereof, and to nucleic acids encoding theantibody and portions thereof (e.g., heavy chain, heavy chain variableregion, light chain, light chain variable region).

[0069] An IgG1 form of mAb 5E4 can be produced by “5E4 CHO stable cellline,” also referred to as CHO 5G4 A1.2C12, which was deposited on Apr.3, 2002, on behalf of Millennium Pharmaceuticals Inc., 75 Sidney Street,Cambridge, Mass., 02139, USA, at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110, U.S.A., under AccessionNo. PTA-4205. The invention relates to cell line CHO 5G4 A1.2C12, to theantibody it produces, antigen-binding fragments thereof, and to nucleicacids encoding the antibody and portions thereof (e.g., heavy chain,heavy chain variable region, light chain, light chain variable region).

[0070] mAb 8D5 can be produced by hybridoma 8D5, also referred to ashybridoma 321 8D5.3.11.8, which was deposited on Apr. 3, 2002, on behalfof Millennium Pharmaceuticals Inc., 75 Sidney Street, Cambridge, Mass.,02139, USA, at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110, U.S.A., under Accession No. PTA-4203.The invention relates to hybridoma 8D5, to the antibody it produces,antigen-binding fragments thereof, and to nucleic acids encoding theantibody and portions thereof (e.g., heavy chain, heavy chain variableregion, light chain, light chain variable region). As described herein,hybridoma 8D5 produces an IgG2 antibody.

[0071] The antibodies and antigen-binding fragments of the invention canbind to the same or similar epitope as mAb 3G6, mAb 5E4 or mAb 8D5.Antibodies and antigen-binding fragments that bind the same or similarepitope as mAb 3G6, mAb 5E4 or mAb 8D5 be identified using any suitablemethod, such as a competitive binding assay. For example, as describedherein, an antibody can be tested for the ability to competitivelyinhibit binding of mAb 3G6, mAb 5E4 or mAb 8D5 to a fusion proteincomprising the I domain of integrin αE chain or to an αE integrin (e.g.,αEβ7) expressed on the surface of a cell. Competitive inhibition ofbinding of mAb 3G6, mAb 5E4 or mAb 8D5 in this type of assay isindicative that the test antibody binds the same or similar epitope asmAb 3G6, mAb 5E4 or mAb 8D5.

[0072] In particular embodiments, the antibody can have the epitopicspecificity of mAb 3G6, mAb 5E4 or mAb 8D5. The fine epitopicspecificity of an antibody can be determined using any suitable method,such as mutational analysis. For example, as described herein, a seriesof integrin αE chain variants comprising amino acid replacements can beprepared and an antibody can be tested for the ability to bind eachvariant. Inhibited or abrogated binding to a variant comprising aparticular amino acid substitution is indicative that the substitutedamino acid is part of the epitope that the antibody binds. (See, Higginset al., J. Biol. Chem. 275:25652-25664 (2000).) In one embodiment, theantibody of the invention has the epitopic specificity of mAb 8D5 andbinds an epitope that comprises Phe298 of integrin αE chain (SEQ ID NO:1).

[0073] In more particular embodiments, the antibody comprises one, twoor three heavy chain complementarity determining regions (HCDR1, HCDR2and/or HCDR3) having the amino acid sequences of the heavy chain CDRs ofmAb 3G6 wherein, optionally, one or two amino acids in each heavy chainCDR can be conservatively substituted, and one, two or three light chaincomplementarity determining regions (LCDR1, LCDR2 and/or LCDR3) havingthe amino acid sequences of the light chain CDRs of mAb 3G6 wherein,optionally, one or two amino acids in each light chain CDR can beconservatively substituted. Preferably, the antibody comprises the threeheavy chain CDRs and the three light chain CDRs of mAb 3G6. In moreparticular embodiments, the antibody comprises the heavy chain variableregion of mAb 3G6 (SEQ ID NO: 4) and the light chain variable region ofmAb 3G6 (SEQ ID NO: 9).

[0074] In other particular embodiments, the antibody comprises one, twoor three heavy chain complementarity determining regions (HCDR1, HCDR2and/or HCDR3) having the amino acid sequences of the heavy chain CDRs ofmAb 5E4 wherein, optionally, one or two amino acids in each heavy chainCDR can be conservatively substituted, and one, two or three light chaincomplementarity determining regions (LCDR1, LCDR2 and/or LCDR3) havingthe amino acid sequences of the light chain CDRs of mAb 5E4 wherein,optionally, one or two amino acids in each light chain CDR can beconservatively substituted. Preferably, the antibody comprises the threeheavy chain CDRs and the three light chain CDRs of mAb 5E4. In moreparticular embodiments, the antibody comprises the heavy chain variableregion of mAb 5E4 (SEQ ID NO: 14) and the light chain variable region ofmAb 5E4 (SEQ ID NO: 19).

[0075] In additional particular embodiments, the antibody comprises one,two or three heavy chain complementarity determining regions (HCDR1,HCDR2 and/or HCDR3) having the amino acid sequences of the heavy chainCDRs of mAb 8D5 wherein, optionally, one or two amino acids in eachheavy chain CDR can be conservatively substituted, and one, two or threelight chain complementarity determining regions (LCDR1, LCDR2 and/orLCDR3) having the amino acid sequences of the light chain CDRs of mAb8D5 wherein, optionally, one or two amino acids in each light chain CDRcan be conservatively substituted. Preferably, the antibody comprisesthe three heavy chain CDRs and the three light chain CDRs of mAb 8D5. Inmore particular embodiments, the antibody comprises the heavy chainvariable region of mAb 8D5 (SEQ ID NO: 24) and the light chain variableregion of mAb 8D5 (SEQ ID NO: 29).

[0076] In additional embodiments, the invention provides novel heavychains and light chains of the antibodies and antigen-binding fragmentsdescribed herein. In particular embodiments, the antibody heavy chainsor antigen-binding portions thereof comprise at least two and preferablythree CDRs having the amino acid sequences of the heavy chain CDRs ofmAb 3G6, the heavy chain CDRs of mAb 5E4 or the heavy chain CDRs of mAb8D5. Optionally, one or two amino acid residues in each heavy chain CDRcan be conservatively substituted. In preferred embodiments, theantibody heavy chains or antigen-binding portions thereof comprise threeCDRs that have the amino acid sequences of the three CDRs of the heavychain of mAb 3G6, the three CDRs of the heavy chain of mAb 5E4 or thethree CDRs of the heavy chain of mAb 8D5. In other embodiments, theantibody heavy chains or antigen-binding portions thereof comprise theheavy chain variable region of mAb 3G6, mAb 5E4 or mAb 8D5. For example,the antibody heavy chains can comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 14 and SEQ ID NO:24. The antibody heavy chains and portions thereof can comprise anysuitable framework regions and/or constant regions (as describedherein).

[0077] In certain embodiments, the antibody light chains orantigen-binding portions thereof comprise at least two and preferablythree CDRs having the amino acid sequences of the light chain CDRs ofmAb 3G6, or the light chain CDRs of mAb 5E4 or the light chain CDRs ofmAb 8D5. Optionally, one or two amino acid residues in each light chainCDR can be conservatively substituted. In preferred embodiments, theantibody light chains or antigen-binding portions thereof comprise threeCDRs that have the amino acid sequences of the three CDRs of the lightchain of mAb 3G6, the three CDRs of the light chain of mAb 5E4 or thethree CDRs of the light chain of mAb 8D5. In other embodiments, theantibody light chains or antigen-binding portions thereof comprise thelight chain variable region of mAb 3G6, mAb 5E4 or mAb 8D5. For example,the antibody light chains can comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 9, SEQ ID NO: 19 and SEQ ID NO:29. The antibody light chains and portions thereof can comprise anysuitable framework regions and/or constant regions (as describedherein).

[0078] Fusion Proteins and Immuno-Conjugates

[0079] Fusion proteins and immunoconjugates can be produced in which anantibody moiety (e.g., antibody or antigen-binding fragment thereof,antibody chain or antigen-binding portion thereof) is linked directly orindirectly to a non-immunoglobulin moiety (i.e., a moiety which does notoccur in immunoglobulins as found in nature). Fusion proteins comprisean antibody moiety and a non-immunoglobulin moiety that are componentsof a single continuous polypeptide chain. The non-immunoglobulin moietycan be located N-terminally, C-terminally or internally with respect tothe antibody moiety. For example, some embodiments can be produced bythe insertion of a nucleic acid encoding immunoglobulin sequences into asuitable expression vector, such as a pET vector (e.g., pET-15b,Novagen), a phage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector(e.g., pRIT2T Protein A fusion vector, Pharmacia). The resultingconstruct can be expressed (e.g., in vivo by a suitable host cell, invitro) to produce antibody chains that comprise a non-immunoglobulinmoiety (e.g., Histidine tag, E tag, Protein A IgG binding domain).Fusion proteins can be isolated or recovered using any suitabletechnique, such as chromatography using a suitable affinity matrix (seee.g., Current Protocols in Molecular Biology (Ausubel, F. M. et al.,eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).

[0080] In other embodiments, the antibody moiety and non-immunoglobulinmoiety may not be part of a continuous polypeptide chain, but can beconnected or conjugated directly or indirectly through any suitablelinker. Suitable methods for connecting or conjugating the moieties arewell known in the art. (See, e.g., Ghetie et al., Pharmacol. Ther.63:209-34 (1994)). A variety of suitable linkers (e.g.,heterobifunctional reagents) and methods for preparing immuno-conjugatesare well known in the art. (See, for example, Hermanson, G. T.,Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).)Suitable non-immunoglobulin moieties for inclusion in animmuno-conjugate include a therapeutic moiety such as a toxin (e.g.,cytotoxin, cytotoxic agent), a therapeutic agent (e.g., achemotherapeutic agent, an antimetabolite, an alkylating agent, ananthracycline, an antibiotic, an anti-mitotic agent, a biologicalresponse modifier (e.g., a cytokine (e.g., an interleukin, aninterferon, a tumor necrosis factor), a growth factor (e.g., aneurotrophic factor)), a plasminogen activator), a radionuclide (e.g, aradioactive ion), an enzyme and the like. Suitable cytotoxins orcytotoxic agents include any agent that is detrimental to cells.Examples of suitable cytotoxins or cytotoxic agents include TAXOL(paclitaxel, Bristol-Myers Squibb Company), cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin (e.g, mitomycin C), etoposide,tenoposide, vincristine, vinblastine, colchicine, doxorubicin,daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, maytansinoids (e.g.,maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos.5,475,092, 5,585,499, 5,846,545), DM1) and analogs or homologs of any ofthe forgoing agents. Suitable therapeutic agents include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepachlorambucil, CC-1065, melphalan,carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine, vinblastine, TAXOL(paclitaxel, Bristol-Myers Squibb Company) and maytansinoids (e.g.,maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos.5,475,092, 5,585,499, 5,846,545), DM1)). Suitable radionuclides include,for example iodine (e.g., iodine-125, -126) yttrium (e.g., yttrium-90,-91) and praseodymium (e.g., praseodymium-144, -145).

[0081] In certain embodiments, the therapeutic agent can be a protein orpolypeptide possessing a desired biological activity. Such proteins orpolypeptides can include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as a tumornecrosis factor (e.g., TNFα, TNFβ), and interferon (e.g., α-interferon,β-interferon, γ-interferion), a neurotrophic factor (e.g., nerve growthfactor), a growth factor (e.g., platelet derived growth factor), aplasminogen activator (e.g., tissue plasminogen activator); orbiological response modifiers such as, for example, cytokines andlymphokines, (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”)), or othergrowth factors. In other embodiments, the antibody or antigen-bindingfragment of the invention can be conjugated to a second antibody orantigen-binding fragment to form an antibody heteroconjugate. (See,e.g., U.S. Pat. No. 4,676,980 (Segal).)

[0082] Nucleic Acids and Constructs

[0083] The present invention also relates to isolated and/or recombinant(including, e.g., essentially pure) nucleic acids comprising sequenceswhich encode an antibody or antigen-binding fragment (e.g., a human,humanized, chimeric antibody or light or heavy chain of any of theforegoing) or fusion protein of the invention.

[0084] Nucleic acids referred to herein as “isolated” are nucleic acidswhich have been separated away from other material (e.g., other nucleicacids such as genomic DNA, cDNA and/or RNA) in its original environment(e.g., in cells or in a mixture of nucleic acids such as a library). Anisolated nucleic acid can be isolated as part of a vector (e.g., aplasmid). Nucleic acids can be naturally occurring, produced by chemicalsynthesis, by combinations of biological and chemical methods (e.g.,semisynthetic), and be isolated using any suitable methods.

[0085] Nucleic acids referred to herein as “recombinant” are nucleicacids which have been produced by recombinant DNA methodology, includingmethods which rely upon artificial recombination, such as cloning into avector or chromosome using, for example, restriction enzymes, homologousrecombination, viruses and the like, and nucleic acids prepared usingthe polymerase chain reaction (PCR). “Recombinant” nucleic acids arealso those that result from recombination of endogenous or exogenousnucleic acids through the natural mechanisms of cells or cells modifiedto allow recombination (e.g., cells modified to express Cre or othersuitable recombinase), but are selected for after the introduction tothe cells of nucleic acids designed to allow and make recombinationprobable. For example, a functionally rearranged human-antibodytransgene is a recombinant nucleic acid.

[0086] The present invention also relates more specifically to nucleicacids that encode the heavy chains and/or light chains of the antibodiesand antigen-binding portions described herein. For example, in oneembodiment, the nucleic acid can encode a heavy chain or antigen-bindingportion thereof that comprises at least one, two or preferably threeCDRs having the amino acid sequences of the heavy chain CDRs of mAb 3G6wherein, optionally, one or two amino acids in each CDR can beconservatively substituted. In another embodiment, the nucleic acid canencode a heavy chain or antigen-binding portion thereof that comprisesat least one, two or preferably three CDRs having the amino acidsequences of the heavy chain CDRs of mAb 5E4 wherein, optionally, one ortwo amino acids in each CDR can be conservatively substituted. Inanother embodiment, the nucleic acid can encode a heavy chain orantigen-binding portion thereof that comprises at least one, two orpreferably three CDRs having the amino acid sequences of the heavy chainCDRs of mAb 8D5 wherein, optionally, one or two amino acids in each CDRcan be conservatively substituted. In preferred embodiments, the nucleicacid encodes an antibody heavy chain or antigen-binding portion thereofthat comprises three CDRs that have the amino acid sequences of thethree CDRs of the heavy chain of mAb 3G6, the three CDRs of the heavychain of mAb 5E4 or the three CDRs of the heavy chain of mAb 8D5. Inother embodiments, the nucleic acid encodes an antibody heavy chain orantigen-binding portion thereof that comprises the heavy chain variableregion of mAb 3G6, mAb 5E4 or mAb 8D5. For example, the nucleic acid cancomprise a nucleotide sequence selected from the group consisting of SEQID NO: 3, SEQ ID NO: 13 and SEQ ID NO: 23. The antibody heavy chains andportions thereof can comprise any suitable framework regions and/orconstant regions (as described herein).

[0087] In another embodiment, the nucleic acid can encode a light chainor antigen-binding portion thereof that comprises at least one, two orpreferably three CDRs having the amino acid sequences of the light chainCDRs of mAb 3G6 wherein, optionally, one or two amino acids in each CDRcan be conservatively substituted. In another embodiment, the nucleicacid can encode a light chain or antigen-binding portion thereof thatcomprises at least one, two or preferably three CDRs having the aminoacid sequences of the light chain CDRs of mAb 5E4 wherein, optionally,one or two amino acids in each CDR can be conservatively substituted. Inanother embodiment, the nucleic acid can encode a light chain orantigen-binding portion thereof that comprises at least one, two orpreferably three CDRs having the amino acid sequences of the light chainCDRs of mAb 8D5 wherein, optionally, one or two amino acids in each CDRcan be conservatively substituted. In preferred embodiments, the nucleicacid encodes an antibody light chain or antigen-binding portion thereofthat comprises three CDRs that have the amino acid sequences of thethree CDRs of the light chain of mAb 3G6, the three CDRs of the lightchain of mAb 5E4 or the three CDRs of the light chain of mAb 8D5. Inother embodiments, the nucleic acid encodes an antibody light chain orantigen-binding portion thereof that comprises the light chain variableregion of mAb 3G6, mAb 5E4 or mAb 8D5. For example, the nucleic acid cancomprise a nucleotide sequence selected from the group consisting of SEQID NO: 8, SEQ ID NO: 18 and SEQ ID NO: 28. The antibody light chains andportions thereof can comprise any suitable framework regions and/orconstant regions (as described herein).

[0088] Nucleic acid molecules of the present invention can be used inthe production of antibodies (e.g., human antibodies, humanizedantibodies, chimeric antibodies and antigen-binding fragments of theforegoing) that bind an αE integrin or integrin αE chain (CD103). Forexample, a nucleic acid (e.g., DNA) encoding an antibody of theinvention can be incorporated into a suitable construct (e.g., anexpression vector) for further manipulation or for production of theencoded polypeptide in suitable host cells.

[0089] Expression constructs or expression vectors suitable for theexpression of a antibody or antigen-binding fragment that binds an αEintegrin are also provided. For example, a nucleic acid encoding all orpart of a desired antibody can be inserted into a nucleic acid vector,such as a plasmid or virus, for expression. The vector can be capable ofreplication in a suitable biological system (e.g., a replicon). Avariety of suitable vectors are known in the art, including vectorswhich are maintained in single copy or multiple copy, or which becomeintegrated into the host cell chromosome.

[0090] Suitable expression vectors can contain a number of components,for example, an origin of replication, a selectable marker gene, one ormore expression control elements, such as a transcription controlelement (e.g., promoter, enhancer, terminator) and/or one or moretranslation signals, a signal sequence or leader sequence, and the like.Expression control elements and a signal or leader sequence, if present,can be provided by the vector or other source. For example, thetranscriptional and/or translational control sequences of a clonednucleic acid encoding an antibody chain can be used to directexpression.

[0091] A promoter can be provided for expression in a desired host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding an antibody, antibodychain or portion thereof, such that it directs transcription of thenucleic acid. A variety of suitable promoters for procaryotic (e.g.,lac, tac, T3, T7 promoters for E. coli) and eucaryotic (e.g., simianvirus 40 early or late promoter, Rous sarcoma virus long terminal repeatpromoter, cytomegalovirus promoter, adenovirus late promoter, EG-1apromoter) hosts are available.

[0092] In addition, expression vectors typically comprise a selectablemarker for selection of host cells carrying the vector, and, in the caseof a replicable expression vector, an origin or replication. Genesencoding products which confer antibiotic or drug resistance are commonselectable markers and may be used in procaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated.

[0093] Suitable expression vectors for expression in mammalian cellsinclude, for example, pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, pEF-1(Invitrogen, Carlsbad, Calif.), pCMV-SCRIPT, pFB, pSG5, pXT1(Stratagene, La Jolla, Calif.), pCDEF3 (Goldman, L. A., et al.,Biotechniques, 21:1013-1015 (1996)), pSVSPORT (GibcoBRL, Rockville,Md.), pEF-Bos (Mizushima, S., et al., Nucleic Acids Res., 18:5322(1990)) and the like. Expression vectors which are suitable for use invarious expression hosts, such as prokaryotic cells (E. coli), insectcells (Drosophila Schnieder S2 cells, Sf9) and yeast (P. methanolica, P.pastoris, S. cerevisiae) are also available.

[0094] Thus, the invention provides an expression vector comprising anucleic acid encoding an antibody, antigen-binding fragment of anantibody (e.g., a human, humanized, chimeric antibody or antigen-bindingfragment of any of the foregoing), antibody chain (e.g., heavy chain,light chain) or antigen-binding portion of an antibody chain that bindsan αE integrin (e.g., an integrin αE chain (CD103)).

[0095] Recombinant Host Cells and Methods of Production

[0096] In another aspect, the invention relates to recombinant hostcells and a method of preparing an antibody or antigen-binding fragment,antibody chain (e.g., heavy chain, light chain) or antigen-bindingportion of an antibody chain, or fusion protein of the invention. Theantibody or antigen-binding fragment can be obtained, for example, bythe expression of one or more recombinant nucleic acids encoding anantibody, antigen-binding fragment of an antibody, antibody chain orantigen-binding portion of an antibody chain that binds an αE integrinin a suitable host cell, or using other suitable methods. For example,the expression constructs described herein can be introduced into asuitable host cell, and the resulting cell can be maintained (e.g., inculture, in an animal, in a plant) under conditions suitable forexpression of the constructs. Suitable host cells can be prokaryotic,including bacterial cells such as E. coli, B. subtilis and/or othersuitable bacteria; eucaryotic cells, such as fungal or yeast cells(e.g., Pichia pastoris, Aspergillus sp., Saccharomyces cerevisiae,Schizosaccharomyces pombe, Neurospora crassa), or other lower eukaryoticcells, and cells of higher eucaryotes such as those from insects (e.g.,Drosophila Schnieder S2 cells, Sf9 insect cells (WO 94/26087(O'Connor)), mammals (e.g., COS cells, such as COS-1 (ATCC Accession No.CRL-1650) and COS-7 (ATCC Accession No. CRL-1651), CHO (e.g., ATCCAccession No. CRL-9096), 293 (ATCC Accession No. CRL-1573), HeLa (ATCCAccession No. CCL-2), CV1 (ATCC Accession No. CCL-70), WOP (Dailey, L.,et al., J. Virol., 54:739-749 (1985), 3T3, 293T (Pear, W. S., et al.,Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396 (1993)) NSO cells, SP2/0,HuT 78 cells and the like, or plants (e.g., tobacco). (See, for example,Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology,Greene Publishing Associates and John Wiley & Sons Inc. (1993).)

[0097] The invention also relates to a recombinant host cell whichcomprises a (one or more) recombinant nucleic acid or expressionconstruct comprising a nucleic acid encoding an antibody,antigen-binding fragment of an antibody (e.g., a human, humanized,chimeric antibody or antigen-binding fragment of any of the foregoing),antibody chain (e.g., heavy chain, light chain) or antigen-bindingportion of an antibody chain that binds an αE integrin (e.g., anintegrin αE chain (CD103)). In particular embodiments, the recombinanthost cell is hybridoma 3G6, hybridoma 5E4, hybridoma 8D5, CHO 3G6 C1.2D6or CHO 5G4 A1.2C12.

[0098] The invention also includes a method of preparing an antibody,antigen-binding fragment of an antibody (e.g., a human, humanized,chimeric antibody or antigen-binding fragment of any of the foregoing),antibody chain (e.g., heavy chain, light chain) or antigen-bindingportion of an antibody chain that binds an αE integrin (e.g., anintegrin αE chain (CD103)), comprising maintaining a recombinant hostcell of the invention under conditions appropriate for expression of anantibody, antigen-binding fragment of an antibody, antibody chain orantigen-binding fragment of an antibody chain. The method can furthercomprise the step of isolating or recovering the antibody,antigen-binding fragment of an antibody, antibody chain orantigen-binding fragment of an antibody chain, if desired.

[0099] For example, a nucleic acid molecule (i.e., one or more nucleicacid molecules) encoding the heavy and light chains of a human antibodythat binds an integrin αE chain, or an expression construct (i.e., oneor more constructs) comprising such nucleic acid molecule(s), can beintroduced into a suitable host cell to create a recombinant host cellusing any method appropriate to the host cell selected (e.g.,transformation, transfection, electroporation, infection), such that thenucleic acid molecule(s) are operably linked to one or more expressioncontrol elements (e.g., in a vector, in a construct created by processesin the cell, integrated into the host cell genome). The resultingrecombinant host cell can be maintained under conditions suitable forexpression (e.g., in the presence of an inducer, in a suitable animal,in suitable culture media supplemented with appropriate salts, growthfactors, antibiotics, nutritional supplements, etc.), whereby theencoded polypeptide(s) are produced. If desired, the encoded protein canbe isolated or recovered (e.g., from the animal, the host cell, medium,milk). This process encompasses expression in a host cell of atransgenic animal (see, e.g., WO 92/03918, GenPharm International).

[0100] The antibodies, antigen-binding fragments, antibody chains andantigen-binding portions thereof described herein can also be producedin a suitable in vitro expression system, by chemical synthesis or byany other suitable method.

[0101] Diagnostic and Therapeutic Methods

[0102] The antibodies (including fragments), fusion proteins andimmuno-conjugates described herein can bind an αE integrin and can beused to detect, measure, select, isolate and/or purify an αE integrin(e.g., αEβ7 integrin) or variants thereof (e.g., by affinitypurification or other suitable methods), and to study αE integrinstructure (e.g., conformation) and function. The antibodies, fusionproteins and immuno-conjugates of the present invention can also be usedin diagnostic applications (e.g., in vitro, ex vivo) and/or intherapeutic applications.

[0103] The antibodies, fusion proteins and immuno-conjugates can be usedto detect and/or measure the level of an αE integrin (e.g., αEβ7integrin) in a sample (e.g., tissues or body fluids, such as aninflammatory exudate, bronchial lavage, blood, serum, bowel fluid,biopsy). In one example, a sample (e.g., tissue and/or body fluid) canbe obtained from an individual and a suitable immunological method canbe used to detect and/or measure αE integrin expression. Suitableimmunological methods for detecting or measuring αE integrin expressioninclude enzyme-linked immunosorbent assays (ELISA), radioimmunoassay,immunohistology, flow cytometry, and the like.

[0104] In one embodiment, the invention is a method of detecting ormeasuring an activated αE integrin in a sample (e.g., a biologicalsample) comprising contacting a sample (e.g., a biological sample) withan antibody or antigen-binding fragment thereof that binds anactivation-induced epitope on an αE integrin (e.g., on an integrin αEchain (CD103)) under conditions suitable for binding of the antibody orantigen-binding fragment to the αE integrin and detecting and/ormeasuring binding of the antibody or antigen-binding fragment to the αEintegrin. Binding of the antibody or antigen-binding fragment thereof tothe αE integrin indicates the presence of the αE integrin in the sample.In an application of the method, an antibody or antigen-binding fragmentof the invention can be used to analyze normal versus inflamed tissues(e.g., from a human) for activated αE integrin reactivity and/orexpression to detect associations between disease (e.g., inflammatorybowel disease, graft rejection) and increased expression of activated αE(e.g., in affected tissues). In embodiments where the antibody orantigen-binding fragment binds an activation-induced epitope, theantibodies, antigen-binding fragments, fusion proteins andimmuno-conjugates of the invention can be used to detect, measure,select, isolate and/or purify activated αE integrin or cells expressingan activated αE integrin.

[0105] The antibodies, fusion proteins and/or immuno-conjugates of thepresent invention permit assessment of the presence of an αE integrin innormal versus inflamed tissues, through which the presence or severityof disease, disease progress and/or the efficacy of therapy can beassessed. For example, therapy can be monitored and efficacy assessed.In one example, an αE integrin can be detected and/or measured in afirst sample obtained from a subject having an inflammatory disease andtherapy can be initiated. Later, a second sample can be obtained fromthe subject and αE integrin in the sample can be detected and/ormeasured. A decrease in the quantity of αE integrin detected or measuredin the second sample can be indicative of therapeutic efficacy.

[0106] The antibodies, fusion proteins and immuno-conjugates describedherein can modulate an activity or function of an αE integrin (e.g.,αEβ7 integrin), such as ligand binding (e.g., E-cadherin) and/orleukocyte infiltration function, including recruitment and/oraccumulation of leukocytes (e.g., T cells) in tissues. Antibodies,fusion proteins and immuno-conjugates that bind an activation-inducedepitope can be used to selectively target cells expressing activated αEintegrin (e.g., αEβ7 integrin) for therapy. For example, an antibodythat binds an activation-induced epitope on an αEβ7 integrin and iscapable of activating complement (e.g., a human IgG1 antibody) can beadministered to selectively deplete cells expressing activated αEβ7through, for example, complement-mediated lysis.

[0107] Preferably the antibodies, fusion proteins and immuno-conjugatescan selectively bind an αE integrin (e.g., αEβ7 integrin) and inhibit αEintegrin-mediated interactions, such as αE integrin-mediated adhesion ofa cell (e.g., T cell) to endothelial cells. In particularly preferredembodiments, the antibodies, fusion proteins and immuno-conjugates caninhibit the interaction of αEβ7 with E-cadherin.

[0108] The antibodies, fusion proteins and immuno-conjugates describedherein can be administered to a subject to modulate an inflammatoryresponse or to treat an inflammatory disease or disorder. For example,an antibody which inhibits the binding of an αE integrin to a ligand(i.e., one or more ligands) can be administered in the treatment ofdiseases associated with leukocyte (e.g., lymphocyte, monocyte)infiltration of tissues, particularly of mucosal tissues. An effectiveamount of an antibody, fusion protein and/or immuno-conjugate (i.e., oneor more) can be administered to a subject (e.g., a mammal, such as ahuman or other primate) in order to treat such a disease. For example,inflammatory diseases, including diseases which are associated withleukocyte infiltration of the gastrointestinal tract (includinggut-associated endothelium), other mucosal tissues, or tissuesexpressing the molecule E-cadherin (e.g., mucosal epithelial surfaces),can be treated according to the present method. Similarly, an individualhaving a disease associated with leukocyte infiltration of tissues as aresult of binding of leukocytes to cells (e.g., epithelial cells)expressing E-cadherin can be treated according to the present invention.

[0109] Examples of inflammatory diseases associated with mucosal tissueswhich can be treated according to the present method include mastitis(mammary gland), cholecystitis, cholangitis or pericholangitis (bileduct and surrounding tissue of the liver), chronic bronchitis, chronicsinusitis, asthma, and graft versus host disease (e.g., in thegastrointestinal tract). As seen in Crohn's disease, mucosalinflammation often extends beyond the mucosal surface. Accordinglychronic inflammatory diseases of the lung which result in interstitialfibrosis, such as hypersensitivity pneumonitis, collagen diseases,sarcoidosis, and other idiopathic conditions can be amenable totreatment.

[0110] According to the method, the severity of symptoms associated withan inflammatory condition can be inhibited (reduced) in whole or inpart. When the subject has a relapsing or chronic condition, aneffective amount of an antibody, fusion protein and/or immuno-conjugateof the invention can be administered to treat the subject, and therapycan be continued (maintenance therapy) with the same or different dosingas indicated, to inhibit relapse or renewed onset of symptoms.Preferably, the antibodies, fusion proteins and/or immuno-conjugates areadministered to treat a subject having a mucosal inflammatory diseases,such as an inflammatory disease of the respiratory tract (e.g.,bronchus, lung), urogenital tract (e.g., kidney, urinary bladder) oralimentary canal and associated organs and tissues (e.g., mouth,salivary glands, esophagus, stomach, small intestine, colon, pancreas,liver, gall bladder).

[0111] In a particularly preferred embodiment, the subject to be treatedhas an inflammatory bowel disease (IBD), such as ulcerative colitis,Crohn's disease, ileitis, Celiac disease, nontropical Sprue, enteropathyassociated with seronegative arthropathies, colitis (e.g., microscopicor collagenous colitis), gastroenteritis (e.g., eosinophilicgastroenteritis), or pouchitis resulting after proctocolectomy andileoanal anastomosis. Subjects having pancreatitis or insulin-dependentdiabetes mellitus can also be treated using the present method. Inanother embodiment, the subject to be treated has an has on oralinflammatory disease, Sjogren's syndrome or Behcet's syndrome.

[0112] In another embodiment, the subject to be treated has a pulmonaryinflammatory disease, such as a chronic obstructive lung disease (e.g.,chronic bronchitis, asthma, silicosis, chronic obstructive pulmonarydisease), hypersensitivity pneumonitis, pulmonary fibrosis (e.g.,idiopathic pulmonary fibrosis) or sarcoidosis. In another embodiment,the subject to be treated has a cutaneous inflammatory disease, such aspsoriasis or inflammatory dermatoses.

[0113] In another embodiment, the invention is a method of inhibitinggraft rejection (e.g., allograft rejection, xenograft rejection) orgraft versus host disease, comprising administering to a subject in needthereof an effective amount of an antibody, fusion protein and/orimmuno-conjugate of the invention. In particular embodiments, thetransplanted graft is a mucosa-associated organ or tissue, such askidney, liver, lung and the like.

[0114] The invention also relates to a method of inhibiting αE integrin(e.g. αEβ7 integrin) mediated homing of leukocytes in a subject,comprising to a subject in need thereof an effective amount of anantibody, fusion protein and/or immuno-conjugate of the invention. Forexample, the homing of leukocytes to mucosal sites (e.g., gut, lung) canbe inhibited.

[0115] As used herein, “subject” refers to humans and animals such asmammals, including, primates, cows, sheep, goats, horses, dogs, cats,rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine,feline, rodent or murine species.

[0116] Diseases and conditions associated with inflammation, infection,and cancer can be treated using the method. In a preferred embodiment,the disease or condition is one in which the actions of cells bearing anαE integrin (e.g., αEβ7), such as lymphocytes (e.g., activated orstimulated T lymphocytes), are to be inhibited or promoted fortherapeutic or prophylactic purposes.

[0117] Diseases or conditions, including chronic diseases, of humans orother species which can be treated with the antibodies, fusion proteinsand/or immuno-conjugates of the invention, include, but are not limitedto:

[0118] inflammatory or allergic diseases and conditions, includingsystemic anaphylaxis or hypersensitivity responses, drug allergies(e.g., to penicillin, cephalosporins), insect sting allergies;inflammatory bowel diseases, such as Crohn's disease, ulcerativecolitis, celiac disease, ileitis and enteritis; sarcoidosis; vaginitis;psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopicdermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g.,necrotizing, cutaneous, and hypersensitivity vasculitis);spondyloarthropathies; scleroderma; respiratory allergic diseases suchas asthma, allergic rhinitis, hypersensitivity lung diseases,hypersensitivity pneumonitis, interstitial lung diseases (ILD) (e.g.,idiopathic pulmonary fibrosis, or ILD associated with rheumatoidarthritis, or other autoimmune conditions);

[0119] autoimmune diseases, such as arthritis (e.g., rheumatoidarthritis, psoriatic arthritis), multiple sclerosis, systemic lupuserythematosus, myasthenia gravis, diabetes, including diabetes mellitusand juvenile onset diabetes, glomerulonephritis and other nephritides,autoimmune thyroiditis, Behcet's syndrome;

[0120] graft rejection (e.g., in transplantation), including allograftrejection or graft-versus-host disease;

[0121] viral infection, e.g., infection by hepatitis C virus (HCV),human papilloma virus (HPV), respiratory syncytial virus, influenzavirus, simian immunodeficiency virus (SIV) or human immunodeficiencyvirus (HIV);

[0122] cancers and/or neoplastic diseases, such as leukemias andlymphomas;

[0123] other diseases or conditions in which undesirable inflammatoryresponses are to be inhibited can be treated, including, but not limitedto, atherosclerosis (e.g., transplant accelerated atherosclerosis),restenosis, cytokine-induced toxicity, myositis (including polymyositis,dermatomyositis).

[0124] Modes of Administration

[0125] According to the method, an (i.e., one or more) antibody,antigen-binding fragment thereof, fusion protein and/or immuno-conjugatecan be administered to the subject by an appropriate route, either aloneor in combination with another drug. An “effective amount” of antibody,fusion protein and/or immuno-conjugate is administered. An “effectiveamount” is an amount sufficient to achieve the desired therapeutic orprophylactic effect, under the conditions of administration, such as anamount sufficient to inhibit binding of αE integrin (αEβ7 integrin) toE-cadherin expressed on epithelial cells, and thereby, inhibit αEintegrin-mediated function, such as leukocyte binding, extravasationand/or retention (e.g., as intra-epithelial lymphocytes (IEL)). Theantibody, fusion protein and/or immuno-conjugate can be administered ina single dose or multiple doses. The antibody or antigen-bindingfragment can be administered as a bolus and/or infusion (e.g.,continuous infusion). The dosage can be determined by methods known inthe art and is dependent, for example, upon the antibody,antigen-binding fragment, fusion protein and/or immuno-conjugate chosen,the subject's age, sensitivity and tolerance to drugs, and overallwell-being. Typically, an effective amount can range from about 0.01 mgper day to about 100 mg per day for an adult. Preferably, the dosageranges from about 1 mg per day to about 100 mg per day or from about 1mg per day to about 10 mg per day. Human, humanized and chimericantibodies can often be administered with less frequency than othertypes of therapeutics. For example, an effective amount of a human,humanized or chimeric antibody (or antigen-binding fragment of any ofthe foregoing) can range from about 0.01 mg/kg to about 5 or 10 mg/kgadministered daily, weekly, biweekly or monthly.

[0126] A variety of routes of administration are possible including, forexample, oral, dietary, topical, transdermal, rectal, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous, intradermal,intraperatoneal injection), and inhalation (e.g., intrabronchial,intranasal or oral inhalation, intranasal drops) routes ofadministration, depending on the agent and disease or condition to betreated. Administration can be local or systemic as indicated. Thepreferred mode of administration can vary depending upon the agentchosen, and the condition (e.g., disease) being treated, however, oralor parenteral administration is generally preferred.

[0127] The antibody, fusion protein and/or immuno-conjugate and anyother therapeutic agent to be administered can be administered as aneutral compound or as a salt. Salts of compounds (e.g., an antibody)containing an amine or other basic group can be obtained, for example,by reacting with a suitable organic or inorganic acid, such as hydrogenchloride, hydrogen bromide, acetic acid, perchloric acid and the like.Compounds with a quaternary ammonium group also contain a counteranionsuch as chloride, bromide, iodide, acetate, perchlorate and the like.Salts of compounds containing a carboxylic acid or other acidicfunctional group can be prepared by reacting with a suitable base, forexample, a hydroxide base. Salts of acidic functional groups contain acountercation such as sodium, potassium and the like.

[0128] The antibody, fusion protein and/or immuno-conjugate can beadministered to the individual as part of a pharmaceutical compositionfor modulation (e.g., inhibition) of αE integrin function (e.g., ligandbinding and/or leukocyte infiltration), or treating a subject having adisease described herein. The pharmaceutical composition can comprise anantibody, antigen-binding fragment, fusion protein and/orimmuno-conjugate of the invention and a pharmaceutically orphysiologically acceptable carrier. Formulation will vary according tothe route of administration selected (e.g., solution, emulsion,capsule). Suitable pharmaceutical and physiological carriers can containinert ingredients which do not interact with the antibody, fusionprotein and/or immuno-conjugate. Standard pharmaceutical formulationtechniques can be employed, such as those described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitablepharmaceutical carriers for parenteral administration include, forexample, sterile water, physiological saline, bacteriostatic saline(saline containing about 0.9% benzyl alcohol), phosphate-bufferedsaline, Hank's solution, Ringer's-lactate and the like. Methods forencapsulating compositions (such as in a coating of hard gelatin orcyclodextran) are known in the art (Baker, et al, “Controlled Release ofBiological Active Agents”, John Wiley and Sons, 1986). For inhalation,the agent can be solubilized and loaded into a suitable dispenser foradministration (e.g., an atomizer, nebulizer or pressurized aerosoldispenser).

[0129] Furthermore, the antibody or fusion protein of the invention andother therapeutic agents that are proteins can be administered via invivo expression of the recombinant protein. In vivo expression can beaccomplished via somatic cell expression according to suitable methods(see, e.g. U.S. Pat. No. 5,399,346). In this embodiment, a nucleic acidencoding the protein can be incorporated into a retroviral, adenoviralor other suitable vector (preferably, a replication deficient infectiousvector) for delivery, or can be introduced into a transfected ortransformed host cell capable of expressing the protein for delivery. Inthe latter embodiment, the cells can be implanted (alone or in a barrierdevice), injected or otherwise introduced in an amount effective toexpress the protein in a therapeutically effective amount.

[0130] The present invention will now be illustrated by the followingExamples, which are not intended to be limiting in any way.

EXAMPLES Methods and Materials

[0131] E-cadherin-IgG Fusion Protein

[0132] A DNA fragment encoding human E-cadherin extracellular domain(residues 1-695 of SEQ ID NO: 34) was isolated by PCR using full-lengthE-cadherin cDNA as template. Synthetic primers Spe-ECAD(5)(gcactagtccaccatgggcccttggagccgc; SEQ ID NO: 42) and ECAD-XHO(3)(ccctcgagaggctgtgccttcctaca; SEQ ID NO: 43) were designed so that SpeIand XhoI restriction sites were incorporated at the 5′ and 3′ of the PCRproduct, respectively. The PCR product was digested with SpeI and XhoI.

[0133] A DNA fragment coding for an human IgG Fc fragment (including thehinge, CH2 and CH3) was isolated by PCR using a fusion construct thatencodes a fusion protein that contains a human IgG1 constant region thathas been mutated to inhibit binding to Fc receptor as template andsynthetic primers Xho-IgG(5) (atctcgagcccaaatcttgtgac; SEQ ID NO: 44)and IgGNot(3) (tagcggccgctcatttacccggagacag; SEQ ID NO: 45) whichintroduced XhoI and NotI sites at the 5′ and 3′ ends of the product,respectively. The product was cut with XhoI and NotI.

[0134] The PCR products (E-cadherin and IgG Fc) were ligated into vectorpCDEF3 (Goldman, L. A., et al., Biotechniques, 21:1013-1015 (1996)) thathad been linearized with SpeI and NotI. Vector pCDEF3 is a derivative ofpcDNA (Invitrogen, Carlsbad, Calif.) and contains the EF-1 promoter. Thesequence of the resulting E-cadherin-IgG fusion construct in pCDEF3 wasconfirmed by DNA sequencing.

[0135] The fusion construct encoded a fusion protein that contained aLeucine residue between the E-cadherin portion and the IgG1 Fc portion,and the IgG1 Fc portion contained mutations to reduce binding to Fcreceptor.

[0136] Expression and Purification

[0137] The E-cadherin-IgG fusion construct was transiently transfectedin 293T cells using calcium phosphate transfection method. 10 μg of theexpression vector was used to transfect one 10 cm plate of 293T cells(Pear, W. S., et al., Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396(1993)). For large scale purification of the fusion protein, 30-35plates of cells were typically transfected. 7-11 hourspost-transfection, the culture medium was changed to media supplementedwith 10% ultra low IgG fetal bovine serum (Gibco). The transfected cellswere cultured and the culture supernatant (10 mL) was collected dailyfor three days. The human IgG was isolated from the collectedsupernatant by chromatography using a protein A column at 4° C. Thecolumn was washed with TBS/Ca (20 mM Tris, pH 7.5, 140 mM NaCl,supplemented with 1 mM CaCl₂), and eluted with 100 mM Glycine-HCl (pH2.3), 1 mM CaCl₂. The eluate was immediately neutralized with 1M Tris pH9.0 (1/15, v/v). Protein fractions were pooled and dialyzed in TBS/Caovernight at 4° C. Protein concentration was determined by the Bradfordmethod (Bio-Rad, Hercules, Calif.) using bovine IgG as standard, andprotein purity was evaluated by SDS-PAGE.

[0138] Biotinylation

[0139] E-cadherin-IgG fusion protein was dialyzed in 10 mM Na-borate (pH8.4), 0.5 mM CaCl₂ for overnight at 4° C., and labeled withaminohexanoyl-biotin-N-hydroxysuccinimide (AH-BNHS, Zymed, South SanFrancisco, Calif.) at a ratio of 1:10 (AH-BNHS/protein, w/w) for 1 hourat room temperature. The labeled protein was dialyzed in TBS (20 mMTris-HCL, pH 7.5, 150 mM NaCl) supplemented with 1 mM CaCl₂ at 4° C.Protein concentration was determined using the Bradford method(Bio-Rad).

[0140] Soluble Recombinant αEβ7 Protein (ts αEβ7.coil)

[0141] A nucleic acid encoding the extracellular domain of integrin αEchain (amino acid residues 1-1105 of SEQ ID NO: 2) was fused with anucleic acid encoding a 30 amino acid acidic peptide(AQLEKELQALEKENAQLEWELQALEKELAQ, SEQ ID NO: 39) to create a constructdesignated αE-acid. A nucleic acid encoding the extracellular domain ofthe β7 subunit (amino acid residues 1-707 of SEQ ID NO: 38) was fusedwith a nucleic acid encoding a 30 amino acid basic peptide(AQLKKKLQALKKKNAQLKWKLQALKKKLAQ, SEQ ID NO: 40) to create a constructdesignated β7-base. When expressed, the acidic and basic peptides form aheterodimeric coiled coil. (See, Lu et al, J. Biol. Chem.276:14642-14648 (2001); O'Shea et al., Curr. Biol. 3:658-667 (1993).) Anucleic acid encoding a linker of six amino acid residues (GGSTGG, SEQID NO: 41) was inserted into both constructs (between the αE sequenceand the acidic peptide, as well as between the β7 and the basicpeptide). The αE and β7 fusion constructs were separately cloned intothe expression vector AprM8 (see, Lu and Springer, J. Immunol.159:268-278 (1997)), and sequences were confirmed by DNA sequencing.

[0142] Expression and Purification

[0143] The αE-acid and β7-base constructs were transiently transfectedinto 293T cells and co-expressed to produce a soluble αEβ7. Thesecretion of soluble αEβ7 heterodimer (ts αEβ7.coil) by the transfectedcells was confirmed by ELISA and immunoprecipitation using severalantibodies that bound the αE or β7 subunit. For large-scalepurification, 30-35 10 cm-plates of 293T cells were co-transfected withαE-acid and β7-base constructs, and culture supernatant was collected asdescribed above.

[0144] ts αEP7.coil was purified by column chromatography using ananti-β7 antibody (mAb 6F7; Millennium Pharmaceuticals Inc., Cambridge,Mass.) affinity column. mAb 6F7 was covalently coupled to CNBr-activatedSEPHAROSE 4B beads (beaded agarose, Pharmacia). Culture supernatantcontaining ts αEβP7.coil was applied to the column at 4° C. The columnwas washed with TBS (20 mM Tris-base, pH 7.5, 150 mM NaCl), 1 mM CaCl₂and 1 mM MgCl₂ in cold, and eluted in 50 mM triethylamine (TEA), pH11.5, 150 mM NaCl, 1 mM CaCl₂ and 1 mM MgCl₂. The eluate was immediatelyneutralized with 1 mM Tris-HCl, pH 6.8, 5 mM CaCl₂ and 5 mM MgCl₂ (1/10,v/v). Protein fractions were pooled, concentrated using CENTRICON-30membrane concentrator (Millipore, Bedford, Mass.), and buffer waschanged to TBS, pH 7.5 containing 1 mM CaCl₂ and 1 mM MgCl₂. The proteinconcentration was determined, and the purified sample was aliquoted andstored at −70° C. The purity of ts αEβ7.coil protein was about 90% asjudged by SDS-PAGE and silver staining.

[0145] Transfectants

[0146] L1.2 cells (murine B lymphoma cell line) were cultured inRPMI/10% FetalClone (Hyclone). K562 cells were maintained in RPMI/10%FBS(Gibco). For stable expression of αEβ7, 20 μg of αE full length cDNA(SEQ ID NO: 1) in AprM8 and 20 μg of β7 cDNA (SEQ ID NO: 37) in AprM8were linearized and cotransfected with 1 μg linear PEFpuro (see, Lu andSpringer, J. Immunol. 159:268-278 (1997)), which contains puromycinselection marker, by electroporation at 250 V, 960 μF using 0.4 cmcuvette. 48 hours post transfection, cells were collected, andresuspended in culture medium supplemented with 2 μg/ml or 4 μg/mlpuromycin for L1.2 transfectants and K562 transfectants, respectively.Cells were subsquently subcloned in 96-well plates. Clones oftransfectants were tested for αEβ7 cell surface expression by stainingwith mouse anti-αE and anti-β7 antibodies. Selected clones weresubcloned again.

[0147] Mouse anti-αE mAb αE7.1 was described previously. (Russel, G. J.et al., Eur. J. Immunol. 24:2832-2841 (1994).) 293T cells (humanembryonic kidney epithelial cell line) were maintained in Dulbecco'sModified Eagles Medium/10% FBS (Gibco), supplemented with essentialamino acids and sodium pyruvate.

[0148] Generation of αEβ7-Specific Human Antibodies

[0149] Human monoclonal antibodies, mAb 3G6, mAb 5E4 and mAb 8D5, weregenerated using human-antibody transgenic mice that express humanimmunoglobulin genes. mAb 5E4 and mAb 3G6 were produced using HUMAB mice(MEDAREX, Princeton, N.J.), and mAb 8D5 was produced using XENOMOUSEmice (Abgenix, Fremont, Calif.). The same immunization, fusion andantibody screening protocols were used to produce human monoclonalantibody 3G6, human monoclonal antibody 5E4 and human monoclonalantibody 8D5.

[0150] Immunization

[0151] L1.2 transfectants that express human αEβ7 were treated withmitomycin C at 100 μg/ml for 30 minutes at 37° C. Cells were washedtwice with phosphate buffered saline (PBS), and resuspended at 2×10⁷cells/ml in PBS. Mice were injected with about 0.5 ml of the resultingcell suspension (intraperitonial injection (IP), 10⁷cells/mouse/injection) at about two week intervals. After 4 IPinjections, mice were boosted with purified recombinant αEβ7 protein (tsαEβ7.coil)(15 μg/mouse, intravenous (IV) injection). 4 days after the IVboost, mice were tested for αEβ7-specific human IgG response in theserum. Spleens from positive mice were used for fusion.

[0152] Titration of αEβ7-Specific Human IgG

[0153] A sandwich ELISA was used to titrate mouse sera containing humanIgG antibodies that bind αEβ7 integrin. ELISA plates were coated with 15μg/ml mouse anti-β7 mAb 6F7 (50 μl/well) at 37° C. for 2 hours. Theplates were then washed with PBS and incubated with 50 μl culturesupernatant containing recombinant αEβ7 protein overnight at 4° C. Theplate was washed twice with PBS, and incubated with mouse anti-serum atvarious dilutions in PBS at 37° C., for 1 hour. Then, the plates werewashed twice, and the plate was incubated with HRP-conjugated goatanti-human IgG at 37° C. for 1 hour. The plates were then washed againand human antibodies that bound αEβ7 were detected by addition ofperoxidase substrate, and absorbance was read on an ELISA reader at 410nM wavelength.

[0154] Hybridomas that Produce Antibodies that Bind αEβ7

[0155] Spleens were removed from mice that produced anti-αEβ7 antibodiesand splenocytes were fused with myeloma cells (SP2/0) to producehybridomas.

[0156] Hybridomas were Screened for Production of Anti-αEβ7 Antibodiesusing a Flow Cytometry Assay and an ELISA.

[0157] L1.2 αEβ7 transfectants or untransfected cells (negative control)were collected by centrifugation, and resuspended to 10⁷ cells/ml inPBS/5% FBS. 50 μl of cell suspension (5×10⁵ cells) was incubated with 50μl hybridoma supernatant in a 96-well plate for 30 minutes on ice. Thecells were washed once with PBS/5% FBS, and incubated withFITC-conjugated anti-human IgG or IgM for 30 minutes on ice. The cellswere washed again, resuspended in PBS, and antibody binding was measuredby flow cytometry using a FACS instrument. Hybridoma supernatants thatstained L1.2 αEβ7 transfectants but not the untransfected parental L1.2cells were saved and screened further by αEβ7-specific ELISA. Theprotocol for the ELISA was identical to the ELISA described above exceptthat 50 μl hybridoma supernatant was used instead of diluted serum.Positive hybridomas were further tested for αE specificity.

[0158] Screen for αE-Specific Antibodies

[0159] FACS staining of K562 transfectants that express either αEβ7 orα4β7 integrin was used. FACs staining protocol was the same as describedabove. Hybridomas that stained αEβ7 transfectants but not α4β7transfectants were selected as producing αE-specific antibody.αE-specific hybridomas were further subcloned at least twice by limitingdilution.

[0160] Assays for Selecting Antibodies that Inhibit Binding of αEβP7 toE-cadherin

[0161] Cell Adhesion Assay.

[0162] ELISA plates were coated with 100 ng/well E-cadherin-IgG fusionprotein in TBS (20 mM Tris, 140 mM NaCl, pH 9)/1 mM CaCl₂ overnight at4° C. Plates were washed with wash buffer (HBSS/1 mM CaCl₂), and blockedwith HBSS/1 mM CaCl₂/2% BSA for one hour at 37° C. After blocking,plates were washed twice with wash buffer. K562 transfectants at loggrowth stage were collected, washed once in HBSS/0.2% BSA/1 mM CaCl₂/1mM MgCl₂, and resuspended to 4×10⁶ cells/mL in the same buffer. Cellswere labeled with the fluorecent dye BCECF-AM (Molecular Probes, 4 μg/mlfinal concentration) for 15 minutes at 3° C. Labeled cells were washedtwice, and resuspended in assay buffer (HBSS/0.2% BSA/1 mM CaCl₂/1 mMMgCl₂/1 mM MnCl₂) to 8×10⁵ cells/mL. 50 μl of the cell suspension wasadded to the E-cadherin-IgG coated well (4×10⁴ cells/well), and mixedwith 50 μl assay buffer containing antibodies with desiredconcentration, or isotype-matched control antibody. The plate was thenincubated at room temperature for 1 hour. The fluorescence content ineach well was read on a Fluorescent Concentration Analyser (IDEXX,Westbrook, Me.) before and after three washes with HBSS/0.5 mM CaCl₂/0.5mM MgCl₂/0.5 mM MnCl₂ using a Microplate Autowasher (Bio-Tekinstruments, Winooski, Vt.). The Microplate Autowasher was programmedwith parameters: 250 μl wash volume, 1× wash cycle, 0 soak time, andaspiration tube depth of 70. The bound cells (after washes) wereexpressed as a percentage of total input cells (before washes) in eachwell. Each sample was set up in triplicate wells.

[0163] The effect of activation of αEβ7 integrin by divalent cations wasevaluated in cell adhesion assays using transfected K562 cells thatexpressed αEβ7 integrin. The transfected K562 cells were fluorescentlylabeled and added to assay wells that were coated with E-cadherin-IgGfusion protein (100 ng/well). The assay media contained CaCl₂ and MgCl₂(1 mM each; Ca+Mg); CaCl₂, MgCl₂ and MnCl₂ (1 mM each; Ca+Mg+Mn); or thedivalent cation chelating agent EDTA (5 mM). The fluorescently labeledcells were allowed to adhere to the plate-bound E-cadherin-IgG fusionprotein, unbound cells were washed away and bound cells are detected bymeasuring fluorescence. Cell binding was enhanced in media thatcontained MnCl₂ and inhibited in media that contained EDTA (relative tomedia that contained media contained CaCl₂ and MgCl₂).

[0164] Cell-Free αEβ7/E-Cadherin Binding Assay.

[0165] Purified recombinant αEβ7 (ts αEβ7.coil) was diluted to 5 μg/mlin TBS, pH 8/Ca+Mg (20 mM Tris, pH 8, 140 mM NaCl, 1 mM CaCl₂ and 1 mMMgCl₂), and 50 μl was used to coat each well of 96-well ELISA plateovernight at 4° C. The plate was washed in wash buffer (20 mM Tris, pH7.5, 140 mM NaCl, 1 mM CaCl₂ and 1 mM MgCl₂), and blocked with 300μl/well blocking buffer (20 mM Tris, pH 7.5, 140 mM NaCl, 1 mM CaCl₂ and1 mM MgCl₂, 2% BSA) for 2 hours at 37° C. 25 μl of biotin-labeledE-cadherin-IgG fusion protein diluted to 20 μg/ml in assay buffer (20 mMTris, pH 7.5, 140 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, and1%BSA) was added to each αEβ7-coated wells, and mixed with 25 μl assaybuffer containing test antibodies at desired concentration, orisotype-matched control antibody. The plate was then incubated for 90minutes at 37° C. The plate was then washed twice with wash buffer, and50 μl HRP-streptavidin (1:1000 dilution in assay buffer) was added toeach well, and the plate was incubated for 1 hour at 37° C. Color wasdeveloped by adding substrate buffer (ABTS substrate for HRP, Zymed),and absorbance was read on an ELISA plate reader (410 nm).

[0166] The effect of activation of αEβ7 integrin by divalent cations wasevaluated in this cell-free adhesion assays using assay buffer thatcontained CaCl₂ and MgCl₂ (1 mM each; Ca+Mg); CaCl₂, MgCl₂ and MnCl₂ (1mM each; Ca+Mg+Mn); or the divalent cation chelating agent EDTA (5 mM).

[0167] Conversion of 5E4 (IgM), 3G6 (IgM) and 8D5 (IgG2) to HumanIgG1-FcRmut Isotype

[0168] RNA was prepared from 1×10⁷ hybridoma cells using QIAGEN RNEASYRNA isolation kit (QIAGEN, Valencia, Calif.) according to manufacturer'sinstruction. cDNA was synthesized, and variable regions of light andheavy chains were cloned out by PCR. VL(kappa) regions were cloned usinghuman IG-PRIMER oligonucleotide primers (Novagen, Madison, Wis.), and VHregions were made using synthetic primers AB85-89 (SEQ ID NOS: 46-50)and AB90 (MEDAREX, Princeton, N.J.; SEQ ID NO: 51) for hybridomas 5E4and 3G6 or synthetic primers pHuVH1-7 (SEQ ID NOS: 52-58) and NHuIgG2p3(SEQ ID NO: 59) for hybridoma 8D5.

[0169] PCR fragments were cloned into PCR2.1-TOPO vector using a TOPOcloning kit (Invitrogen, Carlsbad, Calif.), and 6-8 clones from each PCRreaction were sequenced to determine consensus of variable regionsequences. The variable regions were subsequently isolated fromPCR2.1-TOPO vectors by PCR using primers with restriction enzyme sitesincorporated at both ends for subcloning (MfeI and BlpI sites for VH;EcoRI and BsiWI for 5E4 VL and 3G6 VL; or PpuMI and BsiWI for 8D5 VL).Primers p3G6VH5 (SEQ ID NO: 60) and pAEB7VH3 (SEQ ID NO: 62) were usedfor the 3G6 VH, primers pAEB7VH5 (SEQ ID NO: 61) and pAEB7VH3 (SEQ IDNO: 62) were used for the 5E4 VH, primers pAEB7VK5 (SEQ ID NO: 63) andpAEB7VK3 (SEQ ID NO: 64) were used for the 3G6 and 5E4 VLs. The primersfor the VL of 5E4 and 3G6 include the VL leader sequence whereas allother primers allow cloning into antibody expression vectors thatcontain VH and VL leaders.

[0170] The PCR products encoding the VH of either 5E4 or 3G6 wereseparately subcloned into the MfeI and BlpI sites of pLKTOK30. pLKTOK30is based on the pCDNA3 vector with the CMV promoter replaced with theEF-1a promoter. pLKTOK30 contains sequences encoding a VH leader and ahuman IgG1 constant region that are separated by the desired cloningsites. The human IgG1 constant region encoded by this vector containsthe Leu 235 to Ala 235 and Gly 237 to Ala 237 mutations that interferewith the antibody binding to Fc receptors (human IgG1-FcR mut region).The MfeI site is within the bases VH3-4 and the BlpI site is at thejunction of VH and CH.

[0171] The PCR products encoding the VL of either 5E4 or 3G6 wereseparately subcloned into the EcoRI and BsiWI sites of pLKTOK25.pLKTOK25 has a similar structure to pLKTOK30 with the exception that itcontains a sequence that encodes a human kappa constant region insteadof a human IgG1 constant region and does not contain a sequence encodinga leader. In this vector, the Kozak sequence and sequence encoding a VLleader are included with the adapted VL gene fragments.

[0172] The heavy and light chain containing vectors for each antibody(5E4 or 3G6) were cotransfected in 293T cells to evaluate IgG1production. When production of functional antibody was confirmed, theheavy chain including the promoter region was excised from TOK30 vectorwith HindIII and XbaI and ligated into the same sites (HindIII and XbaI)of the light chain containing TOK25 vector to generate a single IgG1expression vector. The single IgG1 expression vectors were used to makestable CHO cells expressing either 5E4 or 3G6 antibody as describedbelow.

[0173] The VH and VL of 8D5 were adapted and cloned into the antibodyexpression vector pLKTOK59 using PCR. Vector pLKTOK59, like pLKTOK30, isbased on the pCDNA3 vector. However, pLKTOK59 contains two EF-1apromoters, one of which drives expression of the heavy chain while theother drives expression of the light chain. The 8D5 VH gene was adaptedby PCR using synthetic primers p8D5VH5 (SEQ ID NO: 65) and p8D5VH3 (SEQID NO: 66) to add the cloning sites MfeI and BlpI and cloned between theVH leader and Human IgG1-FcRmut region of pLKTOK59D. The 8D5 VL gene wasadapted by PCR using synthetic primers p8D5VK5 (SEQ ID NO: 67) andp8D5VK3 (SEQ ID NO: 68) to add the cloning sites PpuMI and BsiWI andcloned between the VL leader and human kappa constant region ofpLKTOK59D-8D5-VH to create pLKTOK59D-8D5-VHVK.

[0174] Expression of Converted IgG1 Antibodies and Preparation of StableCHO Cells

[0175] Medium scale production of 3G6 (IgG1) and 5E4 (IgG1) was done in293T cells using calcium phosphate transfection. 10 μg of each heavy andlight chain expression vector were used to transfect one 10 cm plate of293T cells. 7-11 hour post-transfection, the culture medium was changedto media supplemented with 10% ultra low IgG FBS (Gibco). Thetransfected cells were cultured and the culture supernatant (10 mL) wascollected daily for three days. A total of about 900 mL supernatant foreach antibody was collected.

[0176] Stable CHO cell lines were generated using the single IgG1expression vectors described above that contain both heavy and lightchains of the converted IgG1 antibodies. CHO (DG44) stable transfectionwas performed using FUGENE non-liposomal lipid transfection (BoehringerMannheim) according to manufacturer's instruction. 2 days aftertransfection, CHO cells were collected, resuspended in selection medium(alphaMEM, 10% Hyclone serum, 800 mg/L G418), and subcloned into 96 wellplates. Several stable CHO clones that secreted IgG1 antibodies wereselected and the high producers were subcloned again. The yield of IgG1production by the stable CHO lines was determined by ELISA assay usinghuman IgG1 as standard, and the functional activity of the IgG1antibodies was determined by binding to αEβ7 transfectants and blockingαEβ7 interaction with E-cadherin.

[0177] The “3G6 CHO stable cell line,” also referred to as CHO 3G6C1.2D6, which produces an IgG1 form of mAb 3G6 was deposited on Apr. 3,2002, on behalf of Millennium Pharmaceuticals Inc., 75 Sidney Street,Cambridge, Mass., 02139, USA, at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110, U.S.A., under AccessionNo. PTA-4204.

[0178] The “5E4 CHO stable cell line,” also referred to as CHO 5G4A1.2C12, which produces an IgG1 form of mAb 5E4 was deposited on Apr. 3,2002, on behalf of Millennium Pharmaceuticals Inc., 75 Sidney Street,Cambridge, Mass., 02139, USA, at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110, U.S.A., under AccessionNo. PTA-4205.

[0179] Purification of IgG Antibodies

[0180] The converted 3G6 (IgG1) and 5E4 (IgG1) antibodies were purifiedfrom culture supernatant of transiently transfected 293T cells, and the8D6 IgG2 antibody was purified from hybridoma supernatant. Protein Aagarose (Gibco) columns were used to purify both IgG1 and IgG2antibodies. Briefly, antibody-containing supernatants were run throughthe Protein A column overnight at 4° C. at a slow flow rate. Then, thecolumn was washed with TBS (20 mM Tris-HCl pH 7.5, 140 mM NaCl) at 4° C.and eluted with 100 mM Glycine-HCl pH 2.3. The eluate was immediatelyneutralized with 1M Tris-HCl pH 9.0 (1/15 v/v). Fractions were pooledand dialyzed in PBS at 4° C. Antibody concentration was determined bythe Bradford method (Bio-Rad) using bovine IgG as standard. Antibodypurity was analyzed by SDS-PAGE.

[0181] Determination of Half Saturation Concentration of mAb Binding toαEβ7 transfctants

[0182] Purified antibodies were serially diluted in PBS/5% FBS. FACSstaining using the diluted antibodies and K562 transfectants thatexpress αEβ7 on the cell surface was performed as described above.FITC-conjugated anti-human IgG1 or FITC-conjugated anti-human IgG2 wasused as secondary antibody. The degree of staining (mean fluorescenceintensity) detected was plotted against the concentration of antibodyused (μg/ml), and half saturation concentrations were determined usingthe plot. To determine Mn²⁺ effect on antibody binding, 1 mM MnCl₂(final concentration) was included in the staining buffer in somestudies.

[0183] Antibody IC50 Determination

[0184] IC50 was determined using the cell adhesion assay and cell-freeαEβ7/E-cadherin binding assays described above. The percentage of inputcells that bound αEβ7 (cell adhesion assay) or amount of E-cadherin-IgGfusion that bound αEβ7 (measured by absorbance in the cell-free bindingassay) was plotted against the concentration of antibody used (μg/ml),an inhibition curve was drawn, and IC50 values were determined using thecurve.

[0185] Culture of Human Peripheral Blood Lymphocytes

[0186] Human PBL were purified from fresh whole blood using standardHistopaque gradient centrifugation, and cultured at 1×10⁶ cells per mLin RPMI 1640 with 10% FBS, supplemented with TGF-β1 and IL-2 to increaseαEβ7 expression on the surface. After culture for 10-15 days, cells werecollected for FACS staining as described above.

[0187] Epitope Mapping

[0188] Construction and Expression of αE I-domain-Fc Fusion Protein.

[0189] A nucleic acid encoding the αE chain I-domain (amino acids161-379 of SEQ ID NO: 2) was isolated from full-length αE cDNA (SEQ IDNO: 1) by PCR using synthetic primers aEXID(5)(tcggatccgctctggagaaggaggag, SEQ ID NO: 69) and aEIDS(3)(gcgaattcaagggcgtctccaaccgt, SEQ ID NO: 70). This nucleic acid wasjoined in-frame with a nucleic acids encoding the αE secretion signalsequence (amino acids −18 to −1 of SEQ ID NO: 2) and a human IgG1 Fcregion that contained mutations to reduce binding to Fc receptor, toproduce a construct encoding the αE I-domain Fc fusion protein.

[0190] The fusion construct encoded a fusion protein that containsGlySer at the amino-terminus of the I domain and GluPhe between the Idomain and the Fc region sequences. The I domain encoded by the fusionconstruct also includes a portion of the X domain. These X domainsequences ensure proper folding and secretion of the I domain fusionprotein.

[0191] The αE I-domain-Fc fusion construct was cloned in vector pCDEF3,which was transiently expressed in 293T cells. Culture supernatant thatcontained the fusion protein was collected as described above.

[0192] Binding of mAb to αE I-domain-Fc Fusion Protein

[0193] An ELISA assay was established to evaluate secretion and foldingof the I-domain fusion protein using mouse antibodies previously mappedto the αE I-domain. (Higgins, J. M. G. et al., J. Biol. Chem.275:25652-25664 (2000).) In this assay, anti-human IgG was immobilizedto capture the αE I-domain-Fc fusion protein from the supernatant of293T transfectants, and binding of two mouse antibodies, αE7.1 andHML-1, was tested. (Higgins, J. M. G. et al., J. Biol. Chem.275:25652-25664 (2000).) The two mouse mAb bound to the αE I-domain-Fcfusion protein. Binding of human antibodies was determined using similarassay conditions. Goat anti-human IgG (15 μg/ml in 20 mM Tris, pH9, 140mM NaCl) was used to coat 96 well ELISA plate overnight at 4° C. Theplate was blocked with 2%BSA and incubated with 50 μl culturesupernatant of 293T transfected with the αE I-domain-Fc fusion constructor with vector alone (control) for 1 hour at 37° C. After washing, theplate was incubated with 50 μl of hybridoma supernatant of 5E4 IgM, 3G6IgM or 8D5 IgG2. Binding of IgM antibody to the captured αE I-domain-Fcfusion protein was detected by HRP-conjugated anti-human IgM oranti-human IgG2.

[0194] Antibody Competition Assay (Cytometry Assay)

[0195] 5×10⁵ K562 transfectants expressing αEβ7 were incubated withmouse mAb αE7.1 (15 μg/ml)(Russel, G. J. et al., Eur. J. Immunol.24:2832-2841 (1994)), human mAb 5E4 (IgM hybridoma supernatant), human3G6 (IgM hybridoma supernatant), or medium control on ice for 30minutes. Then, the cells were washed and incubated with human mAb 8D5(IgG2, 15 μg/ml) for 30 minutes on ice. Cells were then washed twice andincubated with FITC-anti-human IgG, and analyzed by fluorescence flowcytometry.

[0196] Fine Specificity

[0197] The fine specificity of mAb 5E4 was determined using a panel oftransfected K562 cells that expressed various mutant αEβ7 integrins anddetecting antibody binding to the transfectants by flow cytometry. Themutants proteins and methods used have been previously described inHiggins, J. M. G. et al., J. Biol. Chem. 275:25652-25664 (2000). Themutant αEβ7 integrins used contained the following mutations in the αEchain: R159S/R160S; ΔE163-E180; ΔE176; D190A; G193A; D199A; R202A/D205A;G230A/V231A; D240A; F298A; P311H/E345A/T346A; E325A; and Y354W. (See,Higgins, J. M. G. et al., J. Biol. Chem. 275:25652-25664 (2000).)

Results

[0198] Hybridomas that produce human antibodies which bind αEβ7 integrinwere produced, and the antibodies produced by three of the hybridomaswere characterized. The supernatants of hybridomas 3G6 (which producesan IgM), 5E4 (which produces an IgM) and 8D5 (which produces an IgG2)were tested for αEβ7 binding specificity by flow cytometry. mAb 3G6(IgG1), mAb 5E4 (IgG1) and mAb 8D5 each bound transfected L1.2 cells andtransfected K562 cells that expressed αEβ7 integrin, but none of theseantibodies bound transfected K562 cells that expressed α4β7 integrin,indicating that the mAbs have binding specificity for integrin αE chain.Each mAb (mAb 3G6 (IgM), mAb 3G6 (IgG1), mAb 5E4 (IgM), mAb 5E4 (IgG1)and mAb 8D5) inhibited binding of αEβ7 integrin to its ligand E-cadherinusing an in vitro cell adhesion assay and also inhibited binding ofsoluble E-cadherin-Fc to immobilized αEβ7 integrin in a cell freeadhesion assay.

[0199] The variable regions of mAb 3G6, mAb 5E4 and mAb 8D5 were clonedand constructs encoding these antibodies with a human IgG1 constantregion were produced. The IgG1 versions of mAb 3G6 (IgG1) and mAb 5E4(IgG1) were used in some of the studies described herein.

[0200] The concentration of mAb 3G6 (IgG1), mAb 5E4 (IgG1) and mAb 8D5that resulted in half saturation of antibody binding sites ontransfected K562 cells that expressed αEβ7 was determined using flowcytometry. mAb 3G6 (IgG1) and mAb 5E4 (IgG1) both had a half saturationconcentration of 1 μg/mL, while mAb 3G6 had a half saturationconcentration of 2.5 μg/mL. The concentrations of antibody thatinhibited binding in the cell adhesion assay and the cell free bindingassay (IC50) were also determined for mAb 3G6 (IgG1), mAb 5E4 (IgG1) andmAb 8D5. The IC50 for mAb 3G6 (IgG1) was about 2.04 μg/mL (13.4 nM) inthe cell adhesion assay, and about 0.089 μg/mL (0.59 nM) in the cellfree assay. The IC50 for mAb 5E4 (IgG1) was about 1.29 μg/mL (8.5 nM) inthe cell adhesion assay, and about 1.02 μg/mL (6.7 nM) in the cell freeassay. The IC50 for mAb 8D5 (IgG1) was about 0.715 μg/mL (4.7 nM) in thecell adhesion assay, and about 0.197 μg/mL (1.30 nM) in the cell freeassay.

[0201] Integrin molecules, such as αEβ7, bind their ligands with highaffinity when activated by, for example, divalent cations (e.g., Mn²⁺).The results of cellular binding studies revealed that recombinant αEβ7expressed on transfected K562 cells is activated by divalent cationions, particularly Mn²⁺, and binding to immobilized E-cadherin isenhanced under conditions where Mn²⁺ is present. Similar results wereobtained in studies in which transfected K562 cells were stained withBiotin-E-cadherin-IgG. The result of the cellular binding assay arepresented in Table 1, and the results of the staining assay arepresented in Table 2. TABLE 1 Assay Buffer % cells bound to immobilizedE-cadherin Ca²⁺ and Mg²⁺ (1 mM each) ˜40% Ca²⁺ and Mg²⁺ and Mn²⁺ ˜60% (1mM each) EDTA (5 mM) <10%

[0202] TABLE 2 Biotin-E- Binding cadhering-IgG (mean fluorescence used(μg/ml) Staining Buffer intensity) 2.5 Ca²⁺ and Mg²⁺ (1 mM each) <25 Ca²⁺ and Mg²⁺ and Mn²⁺ 50-75 (1 mM each) 5 Ca²⁺ and Mg²⁺ (1 mM each)25-50 Ca²⁺ and Mg²⁺ and Mn²⁺ ˜100 (1 mM each) 10 Ca²⁺ and Mg²⁺ (1 mMeach) ˜75  Ca²⁺ and Mg²⁺ and Mn²⁺ 150-175 (1 mM each)

[0203] To determine whether mAbs 3G6, 5E4 or 8D5 bound anactivation-induced epitope, αEβ7 expressing K562 transfectants werestained with antibodies using a buffer that contained Mn²⁺ and using abuffer that did not contain MW²⁺, and antibody binding was detected byfluorescence flow cytometry. The results of these studies demonstratedthat binding of mAb 3G6 (IgG1) was enhanced in the presence of Mn²⁺, butthat the binding of mAb 5E4 (IgG1), mAb 8D5 and mAb αE7.1 to αEβ7integrin on the K562 transfectants was about equivalent in buffers thatcontained or did not contain Mn²⁺. The results show that mAb 3G6 (IgG1)preferentially bound Mn²⁺ activated αEβ7 integrin on transfected K562cells.

[0204] These results were confirmed in antibody binding studies usingprimary human peripheral blood mononuclear cells. The human PBMC werecultured in IL-2 and TGF-β for 10-15 days, which increased thepercentage of CD3+αE+cells to about 30-40%. The cells were then stainedwith mAb 3G6 (IgG1), mAb 5E4 (IgG1), mAb 8D5 or mAb αE7.1 using a bufferthat contained Mn²⁺ and using a buffer that did not contain Mn²⁺, andantibody binding to CD3+ cells was detected by fluorescence flowcytometry. As with transfected K563 cells, binding of mAb 3G6 (IgG1) wasenhanced in the presence of Mn²⁺ (positive cells in buffer without Mn²⁺,<5%; positive cells in buffer with Mn²⁺, ˜20%), whereas binding of mAb5E4 (IgG1), mAb 8D5 and mAb αE7.1 was about equivalent in buffers thatcontained or did not contain Mn²⁺. These results demonstrate thatintegrin αE chain can adopt an activated conformation and that mAb 3G6preferentially binds an activation-induced epitope on integrin αE chain.

[0205] Epitopic specificity of the mAbs was studied further using an αEI-domain-Fc fusion protein, a panel of transfected K562 cells thatexpressed various mutant αEβ7 integrins (see, Higgins, J. M. et al., J.Biol. Chem. 275:25652-25664 (2000)), and antibody blocking studies usingtransfected K562 cells that expressed αEβ7. Mabs 3G6 (IgM), 5E4 (IgM)and 8D5 (hybridoma culture supernatants) each bound αE I-domain-Fcfusion protein coated wells in the ELISA, but binding above controllevels was not detected in wells coated with supernatants from mocktransfected 293T cells that did not produce the αE I-domain-Fc fusionprotein, demonstrating that each antibody binds an epitope that includesamino acids in the I domain of E-cadherin.

[0206] The fine specificity of mAb 5E4 was examined using transfectedK562 cells that expressed αEβ7 integrin or mutated version of αEβ7integrins, and antibody binding was detected by flow cytometry. Theresults are shown in Table 3. Antibody binding was inhibited by deletionof amino acid residues 163-180 (ΔE163-E180; amino acid residues 163-180of SEQ ID NO: 2), which are in the X domain of integrin αE chain, andwas essentially abrogated by mutation of amino acid residue 298 (aminoacid residue 298 of SEQ ID NO:2), which is in the I-domain, fromPhenylalanine to Alanine (F298A). TABLE 3 % Control % Control αEMutation Staining αE Mutation Staining none 100% R202A/D205A 75-100%R159S/R160S 75-100% G230A/V231A 75-100% ΔE163-E180 ˜25% D240A 75-100%ΔE176 75-100% F298A no binding detected D190A 50-75%  P311H/E345A/T346A50-75%  G193A 75-100% E325A 75-100% D199A 75-100% Y354W ˜75%

[0207] The fine specificity of several anti-αE antibodies have beenevaluated using this method and the mutant αEβ7 integrins. For mostantibodies tested, binding to the ΔE163-E180 mutant is inhibitedrelative to binding to un-mutated αEβ7. Thus, this inhibition appears tobe nonspecific and may be the result of instability of the mutant and/orproteolytic degradation. In contrast, antibody binding was essentiallyabrogated by mutation of amino acid 298, which is in the I-domain, fromPhenylalanine to Alanine (F298A), indicating that epitope bound by mAb5E4 includes Phe298.

[0208] The results of flow cytometry based antibody blocking studiesrevealed that pre-incubating transfected K562 cells that expressed αEβ7integrin with mAb αE7.1 partially inhibited binding of mAb 8D5,indicating that these antibodies may bind adjacent or overlappingepitopes. However, the inhibition could be the result of stericinterference. Binding of mAb 8D5 was not significantly inhibited whenthe transfected cells were pre-incubated with mAb 5E4 (IgM) or mAb 3G6(IgM), demonstrating that mAbs 3G6, 5E4 and 8D5 bind distinct epitopes.

[0209] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

1 70 1 3927 DNA Homo sapiens CDS (126)...(3665) Sig-Peptide (126-179) 1gaattccggc ccccgtgtct gggcgtccgc ctcctggcct cctggctgag gggaagctga 60gtgggccacg gcccatgtgt cgcactcgcc tcggctccca cacagccgcc tctgctccag 120caagg atg tgg ctc ttc cac act ctg ctc tgc ata gcc agc ctg gcc ctg 170Met Trp Leu Phe His Thr Leu Leu Cys Ile Ala Ser Leu Ala Leu 1 5 10 15ctg gcc gct ttc aat gtg gat gtg gcc cgg ccc tgg ctc acg ccc aag 218 LeuAla Ala Phe Asn Val Asp Val Ala Arg Pro Trp Leu Thr Pro Lys 20 25 30 ggaggt gcc cct ttc gtg ctc agc tcc ctt ctg cac caa gac ccc agc 266 Gly GlyAla Pro Phe Val Leu Ser Ser Leu Leu His Gln Asp Pro Ser 35 40 45 acc aaccag acc tgg ctc ctg gtc acc agc ccc aga acc aag agg aca 314 Thr Asn GlnThr Trp Leu Leu Val Thr Ser Pro Arg Thr Lys Arg Thr 50 55 60 cca ggg cccctc cat cga tgt tcc ctt gtc cag gat gaa atc ctt tgc 362 Pro Gly Pro LeuHis Arg Cys Ser Leu Val Gln Asp Glu Ile Leu Cys 65 70 75 cat cct gta gagcat gtc ccc atc ccc aag ggg agg cac cgg gga gtg 410 His Pro Val Glu HisVal Pro Ile Pro Lys Gly Arg His Arg Gly Val 80 85 90 95 acc gtt gtc cggagc cac cac ggt gtt ttg ata tgc att caa gtg ctg 458 Thr Val Val Arg SerHis His Gly Val Leu Ile Cys Ile Gln Val Leu 100 105 110 gtc cgg cgg cctcac agc ctc agc tca gaa ctc aca ggc acc tgt agc 506 Val Arg Arg Pro HisSer Leu Ser Ser Glu Leu Thr Gly Thr Cys Ser 115 120 125 ctc ctg ggc cctgac ctc cgt ccc cag gct cag gcc aac ttc ttc gac 554 Leu Leu Gly Pro AspLeu Arg Pro Gln Ala Gln Ala Asn Phe Phe Asp 130 135 140 ctt gaa aat ctcctg gat cca gat gca cgt gtg gac act gga gac tgc 602 Leu Glu Asn Leu LeuAsp Pro Asp Ala Arg Val Asp Thr Gly Asp Cys 145 150 155 tac agc aac aaagaa ggc ggt gga gaa gac gat gtg aac aca gcc agg 650 Tyr Ser Asn Lys GluGly Gly Gly Glu Asp Asp Val Asn Thr Ala Arg 160 165 170 175 cag cgc cgggct ctg gag aag gag gag gag gaa gac aag gag gag gag 698 Gln Arg Arg AlaLeu Glu Lys Glu Glu Glu Glu Asp Lys Glu Glu Glu 180 185 190 gaa gac gaggag gag gag gaa gct ggc acc gag att gcc atc atc ctg 746 Glu Asp Glu GluGlu Glu Glu Ala Gly Thr Glu Ile Ala Ile Ile Leu 195 200 205 gat ggc tcagga agc att gat ccc cca gac ttt cag aga gcc aaa gac 794 Asp Gly Ser GlySer Ile Asp Pro Pro Asp Phe Gln Arg Ala Lys Asp 210 215 220 ttc atc tccaac atg atg agg aac ttc tat gaa aag tgt ttt gag tgc 842 Phe Ile Ser AsnMet Met Arg Asn Phe Tyr Glu Lys Cys Phe Glu Cys 225 230 235 aac ttt gccttg gtg cag tat gga gga gtg atc cag act gag ttt gac 890 Asn Phe Ala LeuVal Gln Tyr Gly Gly Val Ile Gln Thr Glu Phe Asp 240 245 250 255 ctt cgggac agc cag gat gtg atg gcc tcc ctc gcc aga gtc cag aac 938 Leu Arg AspSer Gln Asp Val Met Ala Ser Leu Ala Arg Val Gln Asn 260 265 270 atc actcaa gtg ggg agt gtc acc aag act gcc tca gcc atg caa cac 986 Ile Thr GlnVal Gly Ser Val Thr Lys Thr Ala Ser Ala Met Gln His 275 280 285 gtc ttagac agc atc ttc acc tca agc cac ggc tcc agg aga aag gca 1034 Val Leu AspSer Ile Phe Thr Ser Ser His Gly Ser Arg Arg Lys Ala 290 295 300 tcc aaggtc atg gtg gtg ctc acc gat ggt ggc ata ttc gag gac ccc 1082 Ser Lys ValMet Val Val Leu Thr Asp Gly Gly Ile Phe Glu Asp Pro 305 310 315 ctc aacctt acg aca gtc atc aac tcc ccc aaa atg cag ggt gtt gag 1130 Leu Asn LeuThr Thr Val Ile Asn Ser Pro Lys Met Gln Gly Val Glu 320 325 330 335 cgcttt gcc att ggg gtg gga gaa gaa ttt aag agt gct agg act gcg 1178 Arg PheAla Ile Gly Val Gly Glu Glu Phe Lys Ser Ala Arg Thr Ala 340 345 350 agggaa ctg aac ctg atc gcc tca gac ccg gat gag acc cat gct ttc 1226 Arg GluLeu Asn Leu Ile Ala Ser Asp Pro Asp Glu Thr His Ala Phe 355 360 365 aaggtg acc aac tac atg gcg ctg gat ggg ctg ctg agc aaa ctg cgg 1274 Lys ValThr Asn Tyr Met Ala Leu Asp Gly Leu Leu Ser Lys Leu Arg 370 375 380 tacaac atc atc agc atg gaa ggc acg gtt gga gac gcc ctt cac tac 1322 Tyr AsnIle Ile Ser Met Glu Gly Thr Val Gly Asp Ala Leu His Tyr 385 390 395 cagctg gca cag att ggc ttc agt gct cag atc ctg gat gag cgg cag 1370 Gln LeuAla Gln Ile Gly Phe Ser Ala Gln Ile Leu Asp Glu Arg Gln 400 405 410 415gtg ctg ctc ggc gcc gtc ggg gcc ttt gac tgg tcc gga ggg gcg ttg 1418 ValLeu Leu Gly Ala Val Gly Ala Phe Asp Trp Ser Gly Gly Ala Leu 420 425 430ctc tac gac aca cgc agc cgc cgg ggc cgc ttc ctg aac cag aca gcg 1466 LeuTyr Asp Thr Arg Ser Arg Arg Gly Arg Phe Leu Asn Gln Thr Ala 435 440 445gcg gcg gcg gca gac gcg gag gct gcg cag tac agc tac ctg ggt tac 1514 AlaAla Ala Ala Asp Ala Glu Ala Ala Gln Tyr Ser Tyr Leu Gly Tyr 450 455 460gct gtg gcc gtg ctg cac aag acc tgc agc ctc tcc tac gtc gcg ggg 1562 AlaVal Ala Val Leu His Lys Thr Cys Ser Leu Ser Tyr Val Ala Gly 465 470 475gct cca cag tac aaa cat cat ggg gcc gtg ttt gag ctc cag aag gag 1610 AlaPro Gln Tyr Lys His His Gly Ala Val Phe Glu Leu Gln Lys Glu 480 485 490495 ggc aga gag gcc agc ttc ctg cca gtg ctg gag gga gag cag atg ggg 1658Gly Arg Glu Ala Ser Phe Leu Pro Val Leu Glu Gly Glu Gln Met Gly 500 505510 tcc tat ttt ggc tct gag ctg tgc cct gtg gac att gac atg gat gga 1706Ser Tyr Phe Gly Ser Glu Leu Cys Pro Val Asp Ile Asp Met Asp Gly 515 520525 agc acg gac ttc ttg ctg gtg gct gct cca ttt tac cac gtt cat gga 1754Ser Thr Asp Phe Leu Leu Val Ala Ala Pro Phe Tyr His Val His Gly 530 535540 gaa gaa ggc aga gtc tac gtg tac cgt ctc agc gag cag gat ggt tct 1802Glu Glu Gly Arg Val Tyr Val Tyr Arg Leu Ser Glu Gln Asp Gly Ser 545 550555 ttc tcc ttg gca cgc ata ctg agt ggg cac ccc ggg ttc acc aat gcc 1850Phe Ser Leu Ala Arg Ile Leu Ser Gly His Pro Gly Phe Thr Asn Ala 560 565570 575 cgc ttt ggc ttt gcc atg gcg gct atg ggg gat ctc agt cag gat aag1898 Arg Phe Gly Phe Ala Met Ala Ala Met Gly Asp Leu Ser Gln Asp Lys 580585 590 ctc aca gat gtg gcc atc ggg gcc ccc ctg gaa ggt ttt ggg gca gat1946 Leu Thr Asp Val Ala Ile Gly Ala Pro Leu Glu Gly Phe Gly Ala Asp 595600 605 gat ggt gcc agc ttc ggc agt gtg tat atc tac aat gga cac tgg gac1994 Asp Gly Ala Ser Phe Gly Ser Val Tyr Ile Tyr Asn Gly His Trp Asp 610615 620 ggc ctc tcc gcc agc ccc tcg cag cgg atc aga gcc tcc acg gtg gcc2042 Gly Leu Ser Ala Ser Pro Ser Gln Arg Ile Arg Ala Ser Thr Val Ala 625630 635 cca gga ctc cag tac ttc ggc atg tcc atg gct ggt ggc ttt gat att2090 Pro Gly Leu Gln Tyr Phe Gly Met Ser Met Ala Gly Gly Phe Asp Ile 640645 650 655 agt ggc gac ggc ctt gcc gac atc acc gtg ggc act ctg ggc caggcg 2138 Ser Gly Asp Gly Leu Ala Asp Ile Thr Val Gly Thr Leu Gly Gln Ala660 665 670 gtt gtg ttc cgc tcc cgg cct gtg gtt cgc ctg aag gtc tcc atggcc 2186 Val Val Phe Arg Ser Arg Pro Val Val Arg Leu Lys Val Ser Met Ala675 680 685 ttc acc ccc agc gca ctg ccc atc ggc ttc aac ggc gtc gtg aatgtc 2234 Phe Thr Pro Ser Ala Leu Pro Ile Gly Phe Asn Gly Val Val Asn Val690 695 700 cgt tta tgt ttt gaa atc agc tct gta acc aca gcc tct gag tcaggc 2282 Arg Leu Cys Phe Glu Ile Ser Ser Val Thr Thr Ala Ser Glu Ser Gly705 710 715 ctc cgt gag gca ctt ctc aac ttc acg ctg gat gtg gat gtg gggaag 2330 Leu Arg Glu Ala Leu Leu Asn Phe Thr Leu Asp Val Asp Val Gly Lys720 725 730 735 cag agg aga cgg ctg cag tgt tca gac gta aga agc tgt ctgggc tgc 2378 Gln Arg Arg Arg Leu Gln Cys Ser Asp Val Arg Ser Cys Leu GlyCys 740 745 750 ctg agg gag tgg agc agc gga tcc cag ctt tgt gag gac ctcctg ctc 2426 Leu Arg Glu Trp Ser Ser Gly Ser Gln Leu Cys Glu Asp Leu LeuLeu 755 760 765 atg ccc aca gag gga gag ctc tgt gag gag gac tgc ttc tccaat gcc 2474 Met Pro Thr Glu Gly Glu Leu Cys Glu Glu Asp Cys Phe Ser AsnAla 770 775 780 agt gtc aaa gtc agc tac cag ctc cag acc cct gag gga cagacg gac 2522 Ser Val Lys Val Ser Tyr Gln Leu Gln Thr Pro Glu Gly Gln ThrAsp 785 790 795 cat ccc cag ccc atc ctg gac cgc tac act gag ccc ttt gccatc ttc 2570 His Pro Gln Pro Ile Leu Asp Arg Tyr Thr Glu Pro Phe Ala IlePhe 800 805 810 815 cag ctg ccc tat gag aag gcc tgc aag aat aag ctg ttttgt gtc gca 2618 Gln Leu Pro Tyr Glu Lys Ala Cys Lys Asn Lys Leu Phe CysVal Ala 820 825 830 gaa tta cag ttg gcc acc acc gtc tct cag cag gag ttggtg gtg ggt 2666 Glu Leu Gln Leu Ala Thr Thr Val Ser Gln Gln Glu Leu ValVal Gly 835 840 845 ctc aca aag gag ctg acc ctg aac att aac cta act aactcc ggg gaa 2714 Leu Thr Lys Glu Leu Thr Leu Asn Ile Asn Leu Thr Asn SerGly Glu 850 855 860 gat tcc tac atg aca agc atg gcc ttg aat tac ccc agaaac ctg cag 2762 Asp Ser Tyr Met Thr Ser Met Ala Leu Asn Tyr Pro Arg AsnLeu Gln 865 870 875 ttg aag agg atg caa aag cct ccc tct cca aac att cagtgt gat gac 2810 Leu Lys Arg Met Gln Lys Pro Pro Ser Pro Asn Ile Gln CysAsp Asp 880 885 890 895 cct cag ccg gtt gct tct gtc ctg atc atg aac tgcagg att ggt cac 2858 Pro Gln Pro Val Ala Ser Val Leu Ile Met Asn Cys ArgIle Gly His 900 905 910 ccc gtc ctc aag agg tca tct gct cat gtt tca gtcgtt tgg cag cta 2906 Pro Val Leu Lys Arg Ser Ser Ala His Val Ser Val ValTrp Gln Leu 915 920 925 gag gag aat gcc ttt cca aac agg aca gca gac atcact gtg act gtc 2954 Glu Glu Asn Ala Phe Pro Asn Arg Thr Ala Asp Ile ThrVal Thr Val 930 935 940 acc aat tcc aat gaa aga cgg tct ttg gcc aac gagacc cac acc ctt 3002 Thr Asn Ser Asn Glu Arg Arg Ser Leu Ala Asn Glu ThrHis Thr Leu 945 950 955 caa ttc agg cat ggc ttc gtt gca gtt ctg tcc aaacca tcc ata atg 3050 Gln Phe Arg His Gly Phe Val Ala Val Leu Ser Lys ProSer Ile Met 960 965 970 975 tac gtg aac aca ggc cag ggg ctt tct cac cacaaa gaa ttc ctc ttc 3098 Tyr Val Asn Thr Gly Gln Gly Leu Ser His His LysGlu Phe Leu Phe 980 985 990 cat gta cat ggg gag aac ctc ttt gga gca gaatac cag ttg caa att 3146 His Val His Gly Glu Asn Leu Phe Gly Ala Glu TyrGln Leu Gln Ile 995 1000 1005 tgc gtc cca acc aaa tta cga ggt ctc caggtt gca gca gtg aag aag 3194 Cys Val Pro Thr Lys Leu Arg Gly Leu Gln ValAla Ala Val Lys Lys 1010 1015 1020 ctg acg agg act cag gcc tcc acg gtgtgc acc tgg agt cag gag cgc 3242 Leu Thr Arg Thr Gln Ala Ser Thr Val CysThr Trp Ser Gln Glu Arg 1025 1030 1035 gct tgt gcg tac agt tcg gtt cagcat gtg gaa gaa tgg cat tca gtg 3290 Ala Cys Ala Tyr Ser Ser Val Gln HisVal Glu Glu Trp His Ser Val 1040 1045 1050 1055 agc tgt gtc atc gct tcagat aaa gaa aat gtc acc gtg gct gca gag 3338 Ser Cys Val Ile Ala Ser AspLys Glu Asn Val Thr Val Ala Ala Glu 1060 1065 1070 atc tcc tgg gat cactct gag gag tta cta aaa gat gta act gaa ctg 3386 Ile Ser Trp Asp His SerGlu Glu Leu Leu Lys Asp Val Thr Glu Leu 1075 1080 1085 cag atc ctt ggtgaa ata tct ttc aac aaa tct cta tat gag gga ctg 3434 Gln Ile Leu Gly GluIle Ser Phe Asn Lys Ser Leu Tyr Glu Gly Leu 1090 1095 1100 aat gca gagaac cac aga act aag atc act gtc gtc ttc ctg aaa gat 3482 Asn Ala Glu AsnHis Arg Thr Lys Ile Thr Val Val Phe Leu Lys Asp 1105 1110 1115 gag aagtac cat tct ttg cct atc atc att aaa ggc agc gtt ggt gga 3530 Glu Lys TyrHis Ser Leu Pro Ile Ile Ile Lys Gly Ser Val Gly Gly 1120 1125 1130 1135ctt ctg gtg ttg atc gtg att ctg gtc atc ctg ttc aag tgt ggc ttt 3578 LeuLeu Val Leu Ile Val Ile Leu Val Ile Leu Phe Lys Cys Gly Phe 1140 11451150 ttt aaa aga aaa tat caa caa ctg aac ttg gag agc atc agg aag gcc3626 Phe Lys Arg Lys Tyr Gln Gln Leu Asn Leu Glu Ser Ile Arg Lys Ala1155 1160 1165 cag ctg aaa tca gag aat ctg ctc gaa gaa gag aat taggacctgctat 3675 Gln Leu Lys Ser Glu Asn Leu Leu Glu Glu Glu Asn * 11701175 ccactgggag aggctatcag ccagtcctgg gacttggaga cccagcatcc tttgcattac3735 tttttccttc aggatgatct agagcagcat ggagctgttg gtagaatatt agtttttaac3795 catacattgt cccaaaagtg tctgtgcatt gtgcaaaaag taaacttagg aaacatttgg3855 tattaaataa atttacactt ttctttgcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa3915 aaaaaaaaaa aa 3927 2 1179 PRT Homo sapiens SIGNAL (1)...(18) 2 MetTrp Leu Phe His Thr Leu Leu Cys Ile Ala Ser Leu Ala Leu Leu -15 -10 -5Ala Ala Phe Asn Val Asp Val Ala Arg Pro Trp Leu Thr Pro Lys Gly 1 5 10Gly Ala Pro Phe Val Leu Ser Ser Leu Leu His Gln Asp Pro Ser Thr 15 20 2530 Asn Gln Thr Trp Leu Leu Val Thr Ser Pro Arg Thr Lys Arg Thr Pro 35 4045 Gly Pro Leu His Arg Cys Ser Leu Val Gln Asp Glu Ile Leu Cys His 50 5560 Pro Val Glu His Val Pro Ile Pro Lys Gly Arg His Arg Gly Val Thr 65 7075 Val Val Arg Ser His His Gly Val Leu Ile Cys Ile Gln Val Leu Val 80 8590 Arg Arg Pro His Ser Leu Ser Ser Glu Leu Thr Gly Thr Cys Ser Leu 95100 105 110 Leu Gly Pro Asp Leu Arg Pro Gln Ala Gln Ala Asn Phe Phe AspLeu 115 120 125 Glu Asn Leu Leu Asp Pro Asp Ala Arg Val Asp Thr Gly AspCys Tyr 130 135 140 Ser Asn Lys Glu Gly Gly Gly Glu Asp Asp Val Asn ThrAla Arg Gln 145 150 155 Arg Arg Ala Leu Glu Lys Glu Glu Glu Glu Asp LysGlu Glu Glu Glu 160 165 170 Asp Glu Glu Glu Glu Glu Ala Gly Thr Glu IleAla Ile Ile Leu Asp 175 180 185 190 Gly Ser Gly Ser Ile Asp Pro Pro AspPhe Gln Arg Ala Lys Asp Phe 195 200 205 Ile Ser Asn Met Met Arg Asn PheTyr Glu Lys Cys Phe Glu Cys Asn 210 215 220 Phe Ala Leu Val Gln Tyr GlyGly Val Ile Gln Thr Glu Phe Asp Leu 225 230 235 Arg Asp Ser Gln Asp ValMet Ala Ser Leu Ala Arg Val Gln Asn Ile 240 245 250 Thr Gln Val Gly SerVal Thr Lys Thr Ala Ser Ala Met Gln His Val 255 260 265 270 Leu Asp SerIle Phe Thr Ser Ser His Gly Ser Arg Arg Lys Ala Ser 275 280 285 Lys ValMet Val Val Leu Thr Asp Gly Gly Ile Phe Glu Asp Pro Leu 290 295 300 AsnLeu Thr Thr Val Ile Asn Ser Pro Lys Met Gln Gly Val Glu Arg 305 310 315Phe Ala Ile Gly Val Gly Glu Glu Phe Lys Ser Ala Arg Thr Ala Arg 320 325330 Glu Leu Asn Leu Ile Ala Ser Asp Pro Asp Glu Thr His Ala Phe Lys 335340 345 350 Val Thr Asn Tyr Met Ala Leu Asp Gly Leu Leu Ser Lys Leu ArgTyr 355 360 365 Asn Ile Ile Ser Met Glu Gly Thr Val Gly Asp Ala Leu HisTyr Gln 370 375 380 Leu Ala Gln Ile Gly Phe Ser Ala Gln Ile Leu Asp GluArg Gln Val 385 390 395 Leu Leu Gly Ala Val Gly Ala Phe Asp Trp Ser GlyGly Ala Leu Leu 400 405 410 Tyr Asp Thr Arg Ser Arg Arg Gly Arg Phe LeuAsn Gln Thr Ala Ala 415 420 425 430 Ala Ala Ala Asp Ala Glu Ala Ala GlnTyr Ser Tyr Leu Gly Tyr Ala 435 440 445 Val Ala Val Leu His Lys Thr CysSer Leu Ser Tyr Val Ala Gly Ala 450 455 460 Pro Gln Tyr Lys His His GlyAla Val Phe Glu Leu Gln Lys Glu Gly 465 470 475 Arg Glu Ala Ser Phe LeuPro Val Leu Glu Gly Glu Gln Met Gly Ser 480 485 490 Tyr Phe Gly Ser GluLeu Cys Pro Val Asp Ile Asp Met Asp Gly Ser 495 500 505 510 Thr Asp PheLeu Leu Val Ala Ala Pro Phe Tyr His Val His Gly Glu 515 520 525 Glu GlyArg Val Tyr Val Tyr Arg Leu Ser Glu Gln Asp Gly Ser Phe 530 535 540 SerLeu Ala Arg Ile Leu Ser Gly His Pro Gly Phe Thr Asn Ala Arg 545 550 555Phe Gly Phe Ala Met Ala Ala Met Gly Asp Leu Ser Gln Asp Lys Leu 560 565570 Thr Asp Val Ala Ile Gly Ala Pro Leu Glu Gly Phe Gly Ala Asp Asp 575580 585 590 Gly Ala Ser Phe Gly Ser Val Tyr Ile Tyr Asn Gly His Trp AspGly 595 600 605 Leu Ser Ala Ser Pro Ser Gln Arg Ile Arg Ala Ser Thr ValAla Pro 610 615 620 Gly Leu Gln Tyr Phe Gly Met Ser Met Ala Gly Gly PheAsp Ile Ser 625 630 635 Gly Asp Gly Leu Ala Asp Ile Thr Val Gly Thr LeuGly Gln Ala Val 640 645 650 Val Phe Arg Ser Arg Pro Val Val Arg Leu LysVal Ser Met Ala Phe 655 660 665 670 Thr Pro Ser Ala Leu Pro Ile Gly PheAsn Gly Val Val Asn Val Arg 675 680 685 Leu Cys Phe Glu Ile Ser Ser ValThr Thr Ala Ser Glu Ser Gly Leu 690 695 700 Arg Glu Ala Leu Leu Asn PheThr Leu Asp Val Asp Val Gly Lys Gln 705 710 715 Arg Arg Arg Leu Gln CysSer Asp Val Arg Ser Cys Leu Gly Cys Leu 720 725 730 Arg Glu Trp Ser SerGly Ser Gln Leu Cys Glu Asp Leu Leu Leu Met 735 740 745 750 Pro Thr GluGly Glu Leu Cys Glu Glu Asp Cys Phe Ser Asn Ala Ser 755 760 765 Val LysVal Ser Tyr Gln Leu Gln Thr Pro Glu Gly Gln Thr Asp His 770 775 780 ProGln Pro Ile Leu Asp Arg Tyr Thr Glu Pro Phe Ala Ile Phe Gln 785 790 795Leu Pro Tyr Glu Lys Ala Cys Lys Asn Lys Leu Phe Cys Val Ala Glu 800 805810 Leu Gln Leu Ala Thr Thr Val Ser Gln Gln Glu Leu Val Val Gly Leu 815820 825 830 Thr Lys Glu Leu Thr Leu Asn Ile Asn Leu Thr Asn Ser Gly GluAsp 835 840 845 Ser Tyr Met Thr Ser Met Ala Leu Asn Tyr Pro Arg Asn LeuGln Leu 850 855 860 Lys Arg Met Gln Lys Pro Pro Ser Pro Asn Ile Gln CysAsp Asp Pro 865 870 875 Gln Pro Val Ala Ser Val Leu Ile Met Asn Cys ArgIle Gly His Pro 880 885 890 Val Leu Lys Arg Ser Ser Ala His Val Ser ValVal Trp Gln Leu Glu 895 900 905 910 Glu Asn Ala Phe Pro Asn Arg Thr AlaAsp Ile Thr Val Thr Val Thr 915 920 925 Asn Ser Asn Glu Arg Arg Ser LeuAla Asn Glu Thr His Thr Leu Gln 930 935 940 Phe Arg His Gly Phe Val AlaVal Leu Ser Lys Pro Ser Ile Met Tyr 945 950 955 Val Asn Thr Gly Gln GlyLeu Ser His His Lys Glu Phe Leu Phe His 960 965 970 Val His Gly Glu AsnLeu Phe Gly Ala Glu Tyr Gln Leu Gln Ile Cys 975 980 985 990 Val Pro ThrLys Leu Arg Gly Leu Gln Val Ala Ala Val Lys Lys Leu 995 1000 1005 ThrArg Thr Gln Ala Ser Thr Val Cys Thr Trp Ser Gln Glu Arg Ala 1010 10151020 Cys Ala Tyr Ser Ser Val Gln His Val Glu Glu Trp His Ser Val Ser1025 1030 1035 Cys Val Ile Ala Ser Asp Lys Glu Asn Val Thr Val Ala AlaGlu Ile 1040 1045 1050 Ser Trp Asp His Ser Glu Glu Leu Leu Lys Asp ValThr Glu Leu Gln 1055 1060 1065 1070 Ile Leu Gly Glu Ile Ser Phe Asn LysSer Leu Tyr Glu Gly Leu Asn 1075 1080 1085 Ala Glu Asn His Arg Thr LysIle Thr Val Val Phe Leu Lys Asp Glu 1090 1095 1100 Lys Tyr His Ser LeuPro Ile Ile Ile Lys Gly Ser Val Gly Gly Leu 1105 1110 1115 Leu Val LeuIle Val Ile Leu Val Ile Leu Phe Lys Cys Gly Phe Phe 1120 1125 1130 LysArg Lys Tyr Gln Gln Leu Asn Leu Glu Ser Ile Arg Lys Ala Gln 1135 11401145 1150 Leu Lys Ser Glu Asn Leu Leu Glu Glu Glu Asn 1155 1160 3 369DNA Homo sapiens 3 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagcccggggagtc tctgaagatc 60 tcctgtaagg gttctggata cagctttacc agctattggatcggctgggt gcgccagatg 120 cccgggaaag gcctggagtg gatggggatc atctatcctggtgactctgg tcccagatac 180 agcccgtcct tccaaggcca ggtcaccatc tcagccgacaagtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgtattactgtgc gcgactgtcg 300 tataccagca cctggtaccc gtactacttt gactactggggccagggaac cctggtcacc 360 gtctcctca 369 4 123 PRT Homo sapiens SITE(31)...(35) CDR1 4 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys LysPro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr SerPhe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys GlyLeu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Gly Pro Arg TyrSer Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser IleSer Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp ThrAla Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Ser Tyr Thr Ser Thr Trp Tyr ProTyr Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val SerSer 115 120 5 5 PRT Homo sapiens 5 Ser Tyr Trp Ile Gly 1 5 6 17 PRT Homosapiens 6 Ile Ile Tyr Pro Gly Asp Ser Gly Pro Arg Tyr Ser Pro Ser PheGln 1 5 10 15 Gly 7 14 PRT Homo sapiens 7 Leu Ser Tyr Thr Ser Thr TrpTyr Pro Tyr Tyr Phe Asp Tyr 1 5 10 8 324 DNA Homo sapiens 8 gaaattgtgttgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgcagggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctcccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180 aggttcagtggcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttgcagtttatta ctgtcagcag cgtagcaact ggcctccggg gacgttcggc 300 caagggaccaaggtggaaat caaa 324 9 108 PRT Homo sapiens SITE (24)...(34) CDR1 9 GluIle Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 7580 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 9095 Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 10 11 PRTHomo sapiens 10 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 11 7PRT Homo sapiens 11 Asp Ala Ser Asn Arg Ala Thr 1 5 12 10 PRT Homosapiens 12 Gln Gln Arg Ser Asn Trp Pro Pro Gly Thr 1 5 10 13 354 DNAHomo sapiens 13 gaggtgcagt tggtggagtc tgggggaggc ttggtccagc ctggagggtccctgagactc 60 tcctgtgcag cctctggatt cacctttagt aacttttgga tgagctgggtccgccaggct 120 ccagggaaag ggctggagtg gatggccaac ataaagcaag atggaagtgagaaatactat 180 gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagagctcactgttt 240 ctgcaaatga acagcctgag agtcgacgac acggctgtat atttctgtgcgggggattac 300 tatgattcgg ggagtttcta ctggggccag ggaaccctgg tcaccgtctcctca 354 14 118 PRT Homo sapiens SITE (31)...(35) CDR1 14 Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Trp MetSer Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ala AsnIle Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys GlyArg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Phe 65 70 75 80 LeuGln Met Asn Ser Leu Arg Val Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 AlaGly Asp Tyr Tyr Asp Ser Gly Ser Phe Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115 15 5 PRT Homo sapiens 15 Asn Phe Trp Met Ser1 5 16 17 PRT Homo sapiens 16 Asn Ile Lys Gln Asp Gly Ser Glu Lys TyrTyr Val Asp Ser Val Lys 1 5 10 15 Gly 17 9 PRT Homo sapiens 17 Asp TyrTyr Asp Ser Gly Ser Phe Tyr 1 5 18 324 DNA Homo sapiens 18 gaaattgtgttgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgcagggccagtca gagtgttagg agcaacttag cctggtacca acagaaacct 120 ggccaggctcccaggctcct catctatgat gcatccaaca gggccattgg catcccagcc 180 aggttcagtggcagtgggtc tgggacagac ttcactctca ccatcagcag tttagagcct 240 gaagattttgtagtttatta ctgtcagcag cgtagcaact ggcctccgtg gacgttcggc 300 caagggaccaaggtggaaat caaa 324 19 108 PRT Homo sapiens SITE (24)...(34) CDR1 19 GluIle Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Ile Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 7580 Glu Asp Phe Val Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 9095 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 20 11 PRTHomo sapiens 20 Arg Ala Ser Gln Ser Val Arg Ser Asn Leu Ala 1 5 10 21 7PRT Homo sapiens 21 Asp Ala Ser Asn Arg Ala Ile 1 5 22 10 PRT Homosapiens 22 Gln Gln Arg Ser Asn Trp Pro Pro Trp Thr 1 5 10 23 384 DNAHomo sapiens 23 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagaccctgtccctc 60 acctgcactg tctctggtgg ctccgtcagt agttactatt ggagctggatccggcagccc 120 ccagggaagg gactggagtg gattggccat atctattaca gtgggaataccaactacaac 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaatcagttctccctg 240 aaactgagct ctgtgaccgc tgcggacacg gccgtgtatt tttgtgcgagagatagatgg 300 aattattatg atagtagtcc cggctattat tattactacg gtatggacgtctggggccaa 360 gggaccacgg tcaccgtcag ctca 384 24 128 PRT Homo sapiensSITE (31)...(35) CDR1 24 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu ValLys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly GlySer Val Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly LysGly Leu Glu Trp Ile 35 40 45 Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn TyrAsn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser LysAsn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Phe Cys Ala 85 90 95 Arg Asp Arg Trp Asn Tyr Tyr Asp Ser SerPro Gly Tyr Tyr Tyr Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 115 120 125 25 5 PRT Homo sapiens 25 Ser TyrTyr Trp Ser 1 5 26 16 PRT Homo sapiens 26 His Ile Tyr Tyr Ser Gly AsnThr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 27 20 PRT Homo sapiens 27Asp Arg Trp Asn Tyr Tyr Asp Ser Ser Pro Gly Tyr Tyr Tyr Tyr Tyr 1 5 1015 Gly Met Asp Val 20 28 321 DNA Homo sapiens 28 gacatccaga tgacccagtctccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcgagtcagggcattaga aatgatttag gctggtatca gcaaaaacca 120 gggaaagccc ctaagcgcctaatctttgct gcatcccatt tgcaaagtgg agtcccttca 180 aggttcagcg gcagtggatctgggacagag ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttattactgtcaacag cataatagtt ccccattcac tttcggccct 300 gggaccagag tggatatcaa a321 29 107 PRT Homo sapiens SITE (24)...(34) CDR1 29 Asp Ile Gln Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val ThrIle Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp TyrGln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Phe Ala Ala SerHis Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser GlyThr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp PheAla Thr Tyr Tyr Cys Gln Gln His Asn Ser Ser Pro Phe 85 90 95 Thr Phe GlyPro Gly Thr Arg Val Asp Ile Lys 100 105 30 11 PRT Homo sapiens 30 ArgAla Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10 31 7 PRT Homo sapiens 31Ala Ala Ser His Leu Gln Ser 1 5 32 9 PRT Homo sapiens 32 Gln Gln His AsnSer Ser Pro Phe Thr 1 5 33 2808 DNA Homo sapiens CDS (109)...(2745) 33ggaaagcacc tgtgagcttg gcaagtcagt tcagagctcc agcccgctcc agcccggccc 60gacccgaccg cacccggcgc ctgcctcgct cgggctcccc ggccagcc atg ggc cct 117 MetGly Pro 1 tgg agc cgc agc ctc tcg ggc ctg ctg ctg ctg ctg agg tct cctctt 165 Trp Ser Arg Ser Leu Ser Gly Leu Leu Leu Leu Leu Arg Ser Pro Leu5 10 15 ggc tct cag gag cgg agc cct cct ccc tgt ttg acg cga gag cta cac213 Gly Ser Gln Glu Arg Ser Pro Pro Pro Cys Leu Thr Arg Glu Leu His 2025 30 35 gtt cac ggt gcc ccg gcg cca cct gag aag agg ccg cgt ctg ggc aga261 Val His Gly Ala Pro Ala Pro Pro Glu Lys Arg Pro Arg Leu Gly Arg 4045 50 gtg aat ttt gaa gat tgc acc ggt cga caa agg aca gct att ttc ctg309 Val Asn Phe Glu Asp Cys Thr Gly Arg Gln Arg Thr Ala Ile Phe Leu 5560 65 aca ccg att ccg aaa gtg ggc aca gat ggt gtg att aca gtc aaa agg357 Thr Pro Ile Pro Lys Val Gly Thr Asp Gly Val Ile Thr Val Lys Arg 7075 80 cct cta cgg ttt cat aac cca aca gat cca ttt ctt ggt cta cgc tgg405 Pro Leu Arg Phe His Asn Pro Thr Asp Pro Phe Leu Gly Leu Arg Trp 8590 95 gac tcc acc tac aga aag ttt tcc acc aaa gtc acg ctg aat aca gtg453 Asp Ser Thr Tyr Arg Lys Phe Ser Thr Lys Val Thr Leu Asn Thr Val 100105 110 115 ggg cac cac cac cgc ccc ccg ccc cat cag gcc tcc gtt tct ggaatc 501 Gly His His His Arg Pro Pro Pro His Gln Ala Ser Val Ser Gly Ile120 125 130 caa gca gaa ttg ctc aca ttt ccc aac tcc tct cct ggc ctc agaaga 549 Gln Ala Glu Leu Leu Thr Phe Pro Asn Ser Ser Pro Gly Leu Arg Arg135 140 145 cag aag aga gac tgg gtt att cct ccc atc agc tgc cca gaa aatgaa 597 Gln Lys Arg Asp Trp Val Ile Pro Pro Ile Ser Cys Pro Glu Asn Glu150 155 160 aaa ggc cca ttt cct aaa aac ctg gtt cag atc aaa tcc aac aaagac 645 Lys Gly Pro Phe Pro Lys Asn Leu Val Gln Ile Lys Ser Asn Lys Asp165 170 175 aaa gaa ggc aag gtt ttc tac agc atc act ggc caa gga gct gacaca 693 Lys Glu Gly Lys Val Phe Tyr Ser Ile Thr Gly Gln Gly Ala Asp Thr180 185 190 195 ccc cct gtt ggt gtc ttt att att gaa aga gaa aca gga tggctg aag 741 Pro Pro Val Gly Val Phe Ile Ile Glu Arg Glu Thr Gly Trp LeuLys 200 205 210 gtg aca gag cct ctg gat aga gaa cgc att gcc aca tac actctc ttc 789 Val Thr Glu Pro Leu Asp Arg Glu Arg Ile Ala Thr Tyr Thr LeuPhe 215 220 225 tct cac gct gtg tca tcc aac ggg aat gca gtt gag gat ccaatg gag 837 Ser His Ala Val Ser Ser Asn Gly Asn Ala Val Glu Asp Pro MetGlu 230 235 240 att ttg atc acg gta acc gat cag aat gac aac aag ccc gaattc acc 885 Ile Leu Ile Thr Val Thr Asp Gln Asn Asp Asn Lys Pro Glu PheThr 245 250 255 cag gag gtc ttt aag ggg tct gtc atg gaa ggt gct ctt ccagga acc 933 Gln Glu Val Phe Lys Gly Ser Val Met Glu Gly Ala Leu Pro GlyThr 260 265 270 275 tct gtg atg gag gtc aca gcc aca gac gcg gac gat gatgtg aac acc 981 Ser Val Met Glu Val Thr Ala Thr Asp Ala Asp Asp Asp ValAsn Thr 280 285 290 tac aat gcc gcc atc gct tac acc atc ctc agc caa gatcct gag ctc 1029 Tyr Asn Ala Ala Ile Ala Tyr Thr Ile Leu Ser Gln Asp ProGlu Leu 295 300 305 cct gac aaa aat atg ttc acc att aac agg aac aca ggagtc atc agt 1077 Pro Asp Lys Asn Met Phe Thr Ile Asn Arg Asn Thr Gly ValIle Ser 310 315 320 gtg gtc acc act ggg ctg gac cga gag agt ttc cct acgtat acc ctg 1125 Val Val Thr Thr Gly Leu Asp Arg Glu Ser Phe Pro Thr TyrThr Leu 325 330 335 gtg gtt caa gct gct gac ctt caa ggt gag ggg tta agcaca aca gca 1173 Val Val Gln Ala Ala Asp Leu Gln Gly Glu Gly Leu Ser ThrThr Ala 340 345 350 355 aca gct gtg atc aca gtc act gac acc aac gat aatcct ccg atc ttc 1221 Thr Ala Val Ile Thr Val Thr Asp Thr Asn Asp Asn ProPro Ile Phe 360 365 370 aat ccc acc acg tac aag ggt cag gtg cct gag aacgag gct aac gtc 1269 Asn Pro Thr Thr Tyr Lys Gly Gln Val Pro Glu Asn GluAla Asn Val 375 380 385 gta atc acc aca ctg aaa gtg act gat gct gat gccccc aat acc cca 1317 Val Ile Thr Thr Leu Lys Val Thr Asp Ala Asp Ala ProAsn Thr Pro 390 395 400 gcg tgg gag gct gta tac acc ata ttg aat gat gatggt gga caa ttt 1365 Ala Trp Glu Ala Val Tyr Thr Ile Leu Asn Asp Asp GlyGly Gln Phe 405 410 415 gtc gtc acc aca aat cca gtg aac aac gat ggc attttg aaa aca gca 1413 Val Val Thr Thr Asn Pro Val Asn Asn Asp Gly Ile LeuLys Thr Ala 420 425 430 435 aag ggc ttg gat ttt gag gcc aag cag cag tacatt cta cac gta gca 1461 Lys Gly Leu Asp Phe Glu Ala Lys Gln Gln Tyr IleLeu His Val Ala 440 445 450 gtg acg aat gtg gta cct ttt gag gtc tct ctcacc acc tcc aca gcc 1509 Val Thr Asn Val Val Pro Phe Glu Val Ser Leu ThrThr Ser Thr Ala 455 460 465 acc gtc acc gtg gat gtg ctg gat gtg aat gaaggc ccc atc ttt gtg 1557 Thr Val Thr Val Asp Val Leu Asp Val Asn Glu GlyPro Ile Phe Val 470 475 480 cct cct gaa aag aga gtg gaa gtg tcc gag gacttt ggc gtg ggc cag 1605 Pro Pro Glu Lys Arg Val Glu Val Ser Glu Asp PheGly Val Gly Gln 485 490 495 gaa atc aca tcc tac act gcc cag gag cca gacaca ttt atg gaa cag 1653 Glu Ile Thr Ser Tyr Thr Ala Gln Glu Pro Asp ThrPhe Met Glu Gln 500 505 510 515 aaa ata aca tat cgg att tgg aga gac actcgc aac tgg ctg gag att 1701 Lys Ile Thr Tyr Arg Ile Trp Arg Asp Thr ArgAsn Trp Leu Glu Ile 520 525 530 aat ccg gac act ggt gcc att tcc act cgggct gag ctg gac agg gag 1749 Asn Pro Asp Thr Gly Ala Ile Ser Thr Arg AlaGlu Leu Asp Arg Glu 535 540 545 gat ttt gag cac gtg aag aac agc acg tacaca gcc cta atc ata gct 1797 Asp Phe Glu His Val Lys Asn Ser Thr Tyr ThrAla Leu Ile Ile Ala 550 555 560 aca gac aat ggt tct cca gtt gct act ggaaca ggg aca ctt ctg ctg 1845 Thr Asp Asn Gly Ser Pro Val Ala Thr Gly ThrGly Thr Leu Leu Leu 565 570 575 atc ctg tct gat gtg aat gac aac gcc cccata cca gaa cct cga act 1893 Ile Leu Ser Asp Val Asn Asp Asn Ala Pro IlePro Glu Pro Arg Thr 580 585 590 595 ata ttc ttc tgt gag agg aat cca aagcct cag gtc ata aac att cat 1941 Ile Phe Phe Cys Glu Arg Asn Pro Lys ProGln Val Ile Asn Ile His 600 605 610 gat gca gac ctt cct ccc aat aca tctccc ttc aca gca gaa cta aca 1989 Asp Ala Asp Leu Pro Pro Asn Thr Ser ProPhe Thr Ala Glu Leu Thr 615 620 625 cac ggg cga gtg ccc aac tgg acc attcag tac aac gac cca acc caa 2037 His Gly Arg Val Pro Asn Trp Thr Ile GlnTyr Asn Asp Pro Thr Gln 630 635 640 gaa tct atc att ttg aag cca aag atggcc tta gag gtg ggt gac tac 2085 Glu Ser Ile Ile Leu Lys Pro Lys Met AlaLeu Glu Val Gly Asp Tyr 645 650 655 aaa atc aat ctc aag ctc atg gat aaccag aat aaa gac caa gtg acc 2133 Lys Ile Asn Leu Lys Leu Met Asp Asn GlnAsn Lys Asp Gln Val Thr 660 665 670 675 acc tta gag gtc agc gtg tgt gactgt gaa ggg gcc gcc ggc gtc tgt 2181 Thr Leu Glu Val Ser Val Cys Asp CysGlu Gly Ala Ala Gly Val Cys 680 685 690 agg aag gca cag cct gtc gaa gcagga ttg caa att cct gcc att ctg 2229 Arg Lys Ala Gln Pro Val Glu Ala GlyLeu Gln Ile Pro Ala Ile Leu 695 700 705 ggg att ctt gga gga att ctt gctttg cta att ctg att ctg ctg ctc 2277 Gly Ile Leu Gly Gly Ile Leu Ala LeuLeu Ile Leu Ile Leu Leu Leu 710 715 720 ttg ctg ttt ctt cgg agg aga gcggtg gtc aaa gag ccc tta ctg ccc 2325 Leu Leu Phe Leu Arg Arg Arg Ala ValVal Lys Glu Pro Leu Leu Pro 725 730 735 cca gag gat gac acc cgg gac aacgtt tat tac tat gat gaa gaa gga 2373 Pro Glu Asp Asp Thr Arg Asp Asn ValTyr Tyr Tyr Asp Glu Glu Gly 740 745 750 755 ggc gga gaa gag gac cag gacttt gac ttg agc cag ctg cac agg ggc 2421 Gly Gly Glu Glu Asp Gln Asp PheAsp Leu Ser Gln Leu His Arg Gly 760 765 770 ctg gac gct cgg cct gaa gtgact cgt aac gac gtt gca cca acc ctc 2469 Leu Asp Ala Arg Pro Glu Val ThrArg Asn Asp Val Ala Pro Thr Leu 775 780 785 atg agt gtc ccc cgg tat cttccc cgc cct gcc aat ccc gat gaa att 2517 Met Ser Val Pro Arg Tyr Leu ProArg Pro Ala Asn Pro Asp Glu Ile 790 795 800 gga aat ttt att gat gaa aatctg aaa gcg gct gat act gac ccc aca 2565 Gly Asn Phe Ile Asp Glu Asn LeuLys Ala Ala Asp Thr Asp Pro Thr 805 810 815 gcc ccg cct tat gat tct ctgctc gtg ttt gac tat gaa gga agc ggt 2613 Ala Pro Pro Tyr Asp Ser Leu LeuVal Phe Asp Tyr Glu Gly Ser Gly 820 825 830 835 tcc gaa gct gct agt ctgagc tcc ctg aac tcc tca gag tca gac aaa 2661 Ser Glu Ala Ala Ser Leu SerSer Leu Asn Ser Ser Glu Ser Asp Lys 840 845 850 gac cag gac tat gac tacttg aac gaa tgg ggc aat ccg ttc aag aag 2709 Asp Gln Asp Tyr Asp Tyr LeuAsn Glu Trp Gly Asn Pro Phe Lys Lys 855 860 865 ctg gct gac atg tac ggaggc ggc gag gac cac tag gggactcgag 2755 Leu Ala Asp Met Tyr Gly Gly GlyGlu Asp His * 870 875 agaggcggcc cagaccatgt gcagaaatgc agaaatcagcgttctggtgt ttt 2808 34 878 PRT Homo sapiens 34 Met Gly Pro Trp Ser ArgSer Leu Ser Gly Leu Leu Leu Leu Leu Arg 1 5 10 15 Ser Pro Leu Gly SerGln Glu Arg Ser Pro Pro Pro Cys Leu Thr Arg 20 25 30 Glu Leu His Val HisGly Ala Pro Ala Pro Pro Glu Lys Arg Pro Arg 35 40 45 Leu Gly Arg Val AsnPhe Glu Asp Cys Thr Gly Arg Gln Arg Thr Ala 50 55 60 Ile Phe Leu Thr ProIle Pro Lys Val Gly Thr Asp Gly Val Ile Thr 65 70 75 80 Val Lys Arg ProLeu Arg Phe His Asn Pro Thr Asp Pro Phe Leu Gly 85 90 95 Leu Arg Trp AspSer Thr Tyr Arg Lys Phe Ser Thr Lys Val Thr Leu 100 105 110 Asn Thr ValGly His His His Arg Pro Pro Pro His Gln Ala Ser Val 115 120 125 Ser GlyIle Gln Ala Glu Leu Leu Thr Phe Pro Asn Ser Ser Pro Gly 130 135 140 LeuArg Arg Gln Lys Arg Asp Trp Val Ile Pro Pro Ile Ser Cys Pro 145 150 155160 Glu Asn Glu Lys Gly Pro Phe Pro Lys Asn Leu Val Gln Ile Lys Ser 165170 175 Asn Lys Asp Lys Glu Gly Lys Val Phe Tyr Ser Ile Thr Gly Gln Gly180 185 190 Ala Asp Thr Pro Pro Val Gly Val Phe Ile Ile Glu Arg Glu ThrGly 195 200 205 Trp Leu Lys Val Thr Glu Pro Leu Asp Arg Glu Arg Ile AlaThr Tyr 210 215 220 Thr Leu Phe Ser His Ala Val Ser Ser Asn Gly Asn AlaVal Glu Asp 225 230 235 240 Pro Met Glu Ile Leu Ile Thr Val Thr Asp GlnAsn Asp Asn Lys Pro 245 250 255 Glu Phe Thr Gln Glu Val Phe Lys Gly SerVal Met Glu Gly Ala Leu 260 265 270 Pro Gly Thr Ser Val Met Glu Val ThrAla Thr Asp Ala Asp Asp Asp 275 280 285 Val Asn Thr Tyr Asn Ala Ala IleAla Tyr Thr Ile Leu Ser Gln Asp 290 295 300 Pro Glu Leu Pro Asp Lys AsnMet Phe Thr Ile Asn Arg Asn Thr Gly 305 310 315 320 Val Ile Ser Val ValThr Thr Gly Leu Asp Arg Glu Ser Phe Pro Thr 325 330 335 Tyr Thr Leu ValVal Gln Ala Ala Asp Leu Gln Gly Glu Gly Leu Ser 340 345 350 Thr Thr AlaThr Ala Val Ile Thr Val Thr Asp Thr Asn Asp Asn Pro 355 360 365 Pro IlePhe Asn Pro Thr Thr Tyr Lys Gly Gln Val Pro Glu Asn Glu 370 375 380 AlaAsn Val Val Ile Thr Thr Leu Lys Val Thr Asp Ala Asp Ala Pro 385 390 395400 Asn Thr Pro Ala Trp Glu Ala Val Tyr Thr Ile Leu Asn Asp Asp Gly 405410 415 Gly Gln Phe Val Val Thr Thr Asn Pro Val Asn Asn Asp Gly Ile Leu420 425 430 Lys Thr Ala Lys Gly Leu Asp Phe Glu Ala Lys Gln Gln Tyr IleLeu 435 440 445 His Val Ala Val Thr Asn Val Val Pro Phe Glu Val Ser LeuThr Thr 450 455 460 Ser Thr Ala Thr Val Thr Val Asp Val Leu Asp Val AsnGlu Gly Pro 465 470 475 480 Ile Phe Val Pro Pro Glu Lys Arg Val Glu ValSer Glu Asp Phe Gly 485 490 495 Val Gly Gln Glu Ile Thr Ser Tyr Thr AlaGln Glu Pro Asp Thr Phe 500 505 510 Met Glu Gln Lys Ile Thr Tyr Arg IleTrp Arg Asp Thr Arg Asn Trp 515 520 525 Leu Glu Ile Asn Pro Asp Thr GlyAla Ile Ser Thr Arg Ala Glu Leu 530 535 540 Asp Arg Glu Asp Phe Glu HisVal Lys Asn Ser Thr Tyr Thr Ala Leu 545 550 555 560 Ile Ile Ala Thr AspAsn Gly Ser Pro Val Ala Thr Gly Thr Gly Thr 565 570 575 Leu Leu Leu IleLeu Ser Asp Val Asn Asp Asn Ala Pro Ile Pro Glu 580 585 590 Pro Arg ThrIle Phe Phe Cys Glu Arg Asn Pro Lys Pro Gln Val Ile 595 600 605 Asn IleHis Asp Ala Asp Leu Pro Pro Asn Thr Ser Pro Phe Thr Ala 610 615 620 GluLeu Thr His Gly Arg Val Pro Asn Trp Thr Ile Gln Tyr Asn Asp 625 630 635640 Pro Thr Gln Glu Ser Ile Ile Leu Lys Pro Lys Met Ala Leu Glu Val 645650 655 Gly Asp Tyr Lys Ile Asn Leu Lys Leu Met Asp Asn Gln Asn Lys Asp660 665 670 Gln Val Thr Thr Leu Glu Val Ser Val Cys Asp Cys Glu Gly AlaAla 675 680 685 Gly Val Cys Arg Lys Ala Gln Pro Val Glu Ala Gly Leu GlnIle Pro 690 695 700 Ala Ile Leu Gly Ile Leu Gly Gly Ile Leu Ala Leu LeuIle Leu Ile 705 710 715 720 Leu Leu Leu Leu Leu Phe Leu Arg Arg Arg AlaVal Val Lys Glu Pro 725 730 735 Leu Leu Pro Pro Glu Asp Asp Thr Arg AspAsn Val Tyr Tyr Tyr Asp 740 745 750 Glu Glu Gly Gly Gly Glu Glu Asp GlnAsp Phe Asp Leu Ser Gln Leu 755 760 765 His Arg Gly Leu Asp Ala Arg ProGlu Val Thr Arg Asn Asp Val Ala 770 775 780 Pro Thr Leu Met Ser Val ProArg Tyr Leu Pro Arg Pro Ala Asn Pro 785 790 795 800 Asp Glu Ile Gly AsnPhe Ile Asp Glu Asn Leu Lys Ala Ala Asp Thr 805 810 815 Asp Pro Thr AlaPro Pro Tyr Asp Ser Leu Leu Val Phe Asp Tyr Glu 820 825 830 Gly Ser GlySer Glu Ala Ala Ser Leu Ser Ser Leu Asn Ser Ser Glu 835 840 845 Ser AspLys Asp Gln Asp Tyr Asp Tyr Leu Asn Glu Trp Gly Asn Pro 850 855 860 PheLys Lys Leu Ala Asp Met Tyr Gly Gly Gly Glu Asp His 865 870 875 35 3567DNA Homo sapiens CDS (411)...(3527) 35 cgccatcccg cgctctgcgg actgggaggcccgggccagg acgcgagtct gcgcagccga 60 ggttccccag cgccccctgc agccgcgcgtaggcagagac ggagcccggc cctgcgcctc 120 cgcaccacgc ccgggacccc acccagcggcccgtacccgg agaagcagcg cgagcacccg 180 aagctcccgg ctcggcggca gaaaccgggagtggggccgg gcgagtgcgc ggcatcccag 240 gccggcccga acgtccgccc gcggtgggccgacttcccct cctcttccct ctctccttcc 300 tttagcccgc tggcgccgga cacgctgcgcctcatctctt ggggcgttct tccccgttgg 360 ccaaccgtcg catcccgtgc aactttggggtagtggccgc ttagtgttga atg ttc 416 Met Phe 1 ccc acc gag agc gca tgg cttggg aag cga ggc gcg aac ccg ggc ccc 464 Pro Thr Glu Ser Ala Trp Leu GlyLys Arg Gly Ala Asn Pro Gly Pro 5 10 15 gaa gcc gcc gtc cgg gag acg gtgatg ctg ttg ctg tgc ctg ggg gtc 512 Glu Ala Ala Val Arg Glu Thr Val MetLeu Leu Leu Cys Leu Gly Val 20 25 30 ccg acc ggc cgc ccc tac aac gtg gacact gag agc gcg ctg ctt tac 560 Pro Thr Gly Arg Pro Tyr Asn Val Asp ThrGlu Ser Ala Leu Leu Tyr 35 40 45 50 cag ggc ccc cac aac acg ctg ttc ggctac tcg gtc gtg ctg cac agc 608 Gln Gly Pro His Asn Thr Leu Phe Gly TyrSer Val Val Leu His Ser 55 60 65 cac ggg gcg aac cga tgg ctc cta gtg ggtgcg ccc act gcc aac tgg 656 His Gly Ala Asn Arg Trp Leu Leu Val Gly AlaPro Thr Ala Asn Trp 70 75 80 ctc gcc aac gct tca gtg atc aat ccc ggg gcgatt tac aga tgc agg 704 Leu Ala Asn Ala Ser Val Ile Asn Pro Gly Ala IleTyr Arg Cys Arg 85 90 95 atc gga aag aat ccc ggc cag acg tgc gaa cag ctccag ctg ggt agc 752 Ile Gly Lys Asn Pro Gly Gln Thr Cys Glu Gln Leu GlnLeu Gly Ser 100 105 110 cct aat gga gaa cct tgt gga aag act tgt ttg gaagag aga gac aat 800 Pro Asn Gly Glu Pro Cys Gly Lys Thr Cys Leu Glu GluArg Asp Asn 115 120 125 130 cag tgg ttg ggg gtc aca ctt tcc aga cag ccagga gaa aat gga tcc 848 Gln Trp Leu Gly Val Thr Leu Ser Arg Gln Pro GlyGlu Asn Gly Ser 135 140 145 atc gtg act tgt ggg cat aga tgg aaa aat atattt tac ata aag aat 896 Ile Val Thr Cys Gly His Arg Trp Lys Asn Ile PheTyr Ile Lys Asn 150 155 160 gaa aat aag ctc ccc act ggt ggt tgc tat ggagtg ccc cct gat tta 944 Glu Asn Lys Leu Pro Thr Gly Gly Cys Tyr Gly ValPro Pro Asp Leu 165 170 175 cga aca gaa ctg agt aaa aga ata gct ccg tgttat caa gat tat gtg 992 Arg Thr Glu Leu Ser Lys Arg Ile Ala Pro Cys TyrGln Asp Tyr Val 180 185 190 aaa aaa ttt gga gaa aat ttt gca tca tgt caagct gga ata tcc agt 1040 Lys Lys Phe Gly Glu Asn Phe Ala Ser Cys Gln AlaGly Ile Ser Ser 195 200 205 210 ttt tac aca aag gat tta att gtg atg ggggcc cca gga tca tct tac 1088 Phe Tyr Thr Lys Asp Leu Ile Val Met Gly AlaPro Gly Ser Ser Tyr 215 220 225 tgg act ggc tct ctt ttt gtc tac aat ataact aca aat aaa tac aag 1136 Trp Thr Gly Ser Leu Phe Val Tyr Asn Ile ThrThr Asn Lys Tyr Lys 230 235 240 gct ttt tta gac aaa caa aat caa gta aaattt gga agt tat tta gga 1184 Ala Phe Leu Asp Lys Gln Asn Gln Val Lys PheGly Ser Tyr Leu Gly 245 250 255 tat tca gtc gga gct ggt cat ttt cgg agccag cat act acc gaa gta 1232 Tyr Ser Val Gly Ala Gly His Phe Arg Ser GlnHis Thr Thr Glu Val 260 265 270 gtc gga gga gct cct caa cat gag cag attggt aag gca tat ata ttc 1280 Val Gly Gly Ala Pro Gln His Glu Gln Ile GlyLys Ala Tyr Ile Phe 275 280 285 290 agc att gat gaa aaa gaa cta aat atctta cat gaa atg aaa ggt aaa 1328 Ser Ile Asp Glu Lys Glu Leu Asn Ile LeuHis Glu Met Lys Gly Lys 295 300 305 aag ctt gga tcg tac ttt gga gct tctgtc tgt gct gtg gac ctc aat 1376 Lys Leu Gly Ser Tyr Phe Gly Ala Ser ValCys Ala Val Asp Leu Asn 310 315 320 gca gat ggc ttc tca gat ctg ctc gtggga gca ccc atg cag agc acc 1424 Ala Asp Gly Phe Ser Asp Leu Leu Val GlyAla Pro Met Gln Ser Thr 325 330 335 atc aga gag gaa gga aga gtg ttt gtgtac atc aac tct ggc tcg gga 1472 Ile Arg Glu Glu Gly Arg Val Phe Val TyrIle Asn Ser Gly Ser Gly 340 345 350 gca gta atg aat gca atg gaa aca aacctc gtt gga agt gac aaa tat 1520 Ala Val Met Asn Ala Met Glu Thr Asn LeuVal Gly Ser Asp Lys Tyr 355 360 365 370 gct gca aga ttt ggg gaa tct atagtt aat ctt ggc gac att gac aat 1568 Ala Ala Arg Phe Gly Glu Ser Ile ValAsn Leu Gly Asp Ile Asp Asn 375 380 385 gat ggc ttt gaa gat gtt gct atcgga gct cca caa gaa gat gac ttg 1616 Asp Gly Phe Glu Asp Val Ala Ile GlyAla Pro Gln Glu Asp Asp Leu 390 395 400 caa ggt gct att tat att tac aatggc cgt gca gat ggg atc tcg tca 1664 Gln Gly Ala Ile Tyr Ile Tyr Asn GlyArg Ala Asp Gly Ile Ser Ser 405 410 415 acc ttc tca cag aga att gaa ggactt cag atc agc aaa tcg tta agt 1712 Thr Phe Ser Gln Arg Ile Glu Gly LeuGln Ile Ser Lys Ser Leu Ser 420 425 430 atg ttt gga cag tct ata tca ggacaa att gat gca gat aat aat ggc 1760 Met Phe Gly Gln Ser Ile Ser Gly GlnIle Asp Ala Asp Asn Asn Gly 435 440 445 450 tat gta gat gta gca gtt ggtgct ttt cgg tct gat tct gct gtc ttg 1808 Tyr Val Asp Val Ala Val Gly AlaPhe Arg Ser Asp Ser Ala Val Leu 455 460 465 cta agg aca aga cct gta gtaatt gtt gac gct tct tta agc cac cct 1856 Leu Arg Thr Arg Pro Val Val IleVal Asp Ala Ser Leu Ser His Pro 470 475 480 gag tca gta aat aga acg aaattt gac tgt gtt gaa aat gga tgg cct 1904 Glu Ser Val Asn Arg Thr Lys PheAsp Cys Val Glu Asn Gly Trp Pro 485 490 495 tct gtg tgc ata gat cta acactt tgt ttc tca tat aag ggc aag gaa 1952 Ser Val Cys Ile Asp Leu Thr LeuCys Phe Ser Tyr Lys Gly Lys Glu 500 505 510 gtt cca ggt tac att gtt ttgttt tat aac atg agt ttg gat gtg aac 2000 Val Pro Gly Tyr Ile Val Leu PheTyr Asn Met Ser Leu Asp Val Asn 515 520 525 530 aga aag gca gag tct ccacca aga ttc tat ttc tct tct aat gga act 2048 Arg Lys Ala Glu Ser Pro ProArg Phe Tyr Phe Ser Ser Asn Gly Thr 535 540 545 tct gac gtg att aca ggaagc ata cag gtg tcc agc aga gaa gct aac 2096 Ser Asp Val Ile Thr Gly SerIle Gln Val Ser Ser Arg Glu Ala Asn 550 555 560 tgt aga aca cat caa gcattt atg cgg aaa gat gtg cgg gac atc ctc 2144 Cys Arg Thr His Gln Ala PheMet Arg Lys Asp Val Arg Asp Ile Leu 565 570 575 acc cca att cag att gaagct gct tac cac ctt ggt cct cat gtc atc 2192 Thr Pro Ile Gln Ile Glu AlaAla Tyr His Leu Gly Pro His Val Ile 580 585 590 agt aaa cga agt aca gaggaa ttc cca cca ctt cag cca att ctt cag 2240 Ser Lys Arg Ser Thr Glu GluPhe Pro Pro Leu Gln Pro Ile Leu Gln 595 600 605 610 cag aag aaa gaa aaagac ata atg aaa aaa aca ata aac ttt gca agg 2288 Gln Lys Lys Glu Lys AspIle Met Lys Lys Thr Ile Asn Phe Ala Arg 615 620 625 ttt tgt gcc cat gaaaat tgt tct gct gat tta cag gtt tct gca aag 2336 Phe Cys Ala His Glu AsnCys Ser Ala Asp Leu Gln Val Ser Ala Lys 630 635 640 att ggg ttt ttg aagccc cat gaa aat aaa aca tat ctt gct gtt ggg 2384 Ile Gly Phe Leu Lys ProHis Glu Asn Lys Thr Tyr Leu Ala Val Gly 645 650 655 agt atg aag aca ttgatg ttg aat gtg tcc ttg ttt aat gct gga gat 2432 Ser Met Lys Thr Leu MetLeu Asn Val Ser Leu Phe Asn Ala Gly Asp 660 665 670 gat gca tat gaa acgact cta cat gtc aaa cta ccc gtg ggt ctt tat 2480 Asp Ala Tyr Glu Thr ThrLeu His Val Lys Leu Pro Val Gly Leu Tyr 675 680 685 690 ttc att aag atttta gag ctg gaa gag aag caa ata aac tgt gaa gtc 2528 Phe Ile Lys Ile LeuGlu Leu Glu Glu Lys Gln Ile Asn Cys Glu Val 695 700 705 aca gat aac tctggc gtg gta caa ctt gac tgc agt att ggc tat ata 2576 Thr Asp Asn Ser GlyVal Val Gln Leu Asp Cys Ser Ile Gly Tyr Ile 710 715 720 tat gta gat catctc tca agg ata gat att agc ttt ctc ctg gat gtg 2624 Tyr Val Asp His LeuSer Arg Ile Asp Ile Ser Phe Leu Leu Asp Val 725 730 735 agc tca ctc agcaga gcg gaa gag gac ctc agt atc aca gtg cat gct 2672 Ser Ser Leu Ser ArgAla Glu Glu Asp Leu Ser Ile Thr Val His Ala 740 745 750 acc tgt gaa aatgaa gag gaa atg gac aat cta aag cac agc aga gtg 2720 Thr Cys Glu Asn GluGlu Glu Met Asp Asn Leu Lys His Ser Arg Val 755 760 765 770 act gta gcaata cct tta aaa tat gag gtt aag ctg act gtt cat ggg 2768 Thr Val Ala IlePro Leu Lys Tyr Glu Val Lys Leu Thr Val His Gly 775 780 785 ttt gta aaccca act tca ttt gtg tat gga tca aat gat gaa aat gag 2816 Phe Val Asn ProThr Ser Phe Val Tyr Gly Ser Asn Asp Glu Asn Glu 790 795 800 cct gaa acgtgc atg gtg gag aaa atg aac tta act ttc cat gtt atc 2864 Pro Glu Thr CysMet Val Glu Lys Met Asn Leu Thr Phe His Val Ile 805 810 815 aac act ggcaat agt atg gct ccc aat gtt agt gtg gaa ata atg gta 2912 Asn Thr Gly AsnSer Met Ala Pro Asn Val Ser Val Glu Ile Met Val 820 825 830 cca aat tctttt agc ccc caa act gat aag ctg ttc aac att ttg gat 2960 Pro Asn Ser PheSer Pro Gln Thr Asp Lys Leu Phe Asn Ile Leu Asp 835 840 845 850 gtc cagact act act gga gaa tgc cac ttt gaa aat tat caa aga gtg 3008 Val Gln ThrThr Thr Gly Glu Cys His Phe Glu Asn Tyr Gln Arg Val 855 860 865 tgt gcatta gag cag caa aag agt gca atg cag acc ttg aaa ggc ata 3056 Cys Ala LeuGlu Gln Gln Lys Ser Ala Met Gln Thr Leu Lys Gly Ile 870 875 880 gtc cagttc ttg tcc aag act gat aag agg cta ttg tac tgc ata aaa 3104 Val Gln PheLeu Ser Lys Thr Asp Lys Arg Leu Leu Tyr Cys Ile Lys 885 890 895 gct gatcca cat tgt tta aat ttc ttg tgt aat ttt ggg aaa atg gaa 3152 Ala Asp ProHis Cys Leu Asn Phe Leu Cys Asn Phe Gly Lys Met Glu 900 905 910 agt ggaaaa gaa gcc agt gtt cat atc caa ctg gaa ggc cgg cca tcc 3200 Ser Gly LysGlu Ala Ser Val His Ile Gln Leu Glu Gly Arg Pro Ser 915 920 925 930 atttta gaa atg gat gag act tca gca ctc aag ttt gaa ata aga gca 3248 Ile LeuGlu Met Asp Glu Thr Ser Ala Leu Lys Phe Glu Ile Arg Ala 935 940 945 acaggt ttt cca gag cca aat cca aga gta att gaa cta aac aag gat 3296 Thr GlyPhe Pro Glu Pro Asn Pro Arg Val Ile Glu Leu Asn Lys Asp 950 955 960 gagaat gtt gcg cat gtt cta ctg gaa gga cta cat cat caa aga ccc 3344 Glu AsnVal Ala His Val Leu Leu Glu Gly Leu His His Gln Arg Pro 965 970 975 aaacgt tat ttc acc ata gtg att att tca agt agc ttg cta ctt gga 3392 Lys ArgTyr Phe Thr Ile Val Ile Ile Ser Ser Ser Leu Leu Leu Gly 980 985 990 cttatt gta ctt ctg ttg atc tca tat gtt atg tgg aag gct ggc ttc 3440 Leu IleVal Leu Leu Leu Ile Ser Tyr Val Met Trp Lys Ala Gly Phe 995 1000 10051010 ttt aaa aga caa tac aaa tct atc cta caa gaa gaa aac aga aga gac3488 Phe Lys Arg Gln Tyr Lys Ser Ile Leu Gln Glu Glu Asn Arg Arg Asp1015 1020 1025 agt tgg agt tat atc aac agt aaa agc aat gat gat taaggacttcttt 3537 Ser Trp Ser Tyr Ile Asn Ser Lys Ser Asn Asp Asp * 10301035 caaattgaga gaatggaaaa cagcccgccc 3567 36 1038 PRT Homo sapiens 36Met Phe Pro Thr Glu Ser Ala Trp Leu Gly Lys Arg Gly Ala Asn Pro 1 5 1015 Gly Pro Glu Ala Ala Val Arg Glu Thr Val Met Leu Leu Leu Cys Leu 20 2530 Gly Val Pro Thr Gly Arg Pro Tyr Asn Val Asp Thr Glu Ser Ala Leu 35 4045 Leu Tyr Gln Gly Pro His Asn Thr Leu Phe Gly Tyr Ser Val Val Leu 50 5560 His Ser His Gly Ala Asn Arg Trp Leu Leu Val Gly Ala Pro Thr Ala 65 7075 80 Asn Trp Leu Ala Asn Ala Ser Val Ile Asn Pro Gly Ala Ile Tyr Arg 8590 95 Cys Arg Ile Gly Lys Asn Pro Gly Gln Thr Cys Glu Gln Leu Gln Leu100 105 110 Gly Ser Pro Asn Gly Glu Pro Cys Gly Lys Thr Cys Leu Glu GluArg 115 120 125 Asp Asn Gln Trp Leu Gly Val Thr Leu Ser Arg Gln Pro GlyGlu Asn 130 135 140 Gly Ser Ile Val Thr Cys Gly His Arg Trp Lys Asn IlePhe Tyr Ile 145 150 155 160 Lys Asn Glu Asn Lys Leu Pro Thr Gly Gly CysTyr Gly Val Pro Pro 165 170 175 Asp Leu Arg Thr Glu Leu Ser Lys Arg IleAla Pro Cys Tyr Gln Asp 180 185 190 Tyr Val Lys Lys Phe Gly Glu Asn PheAla Ser Cys Gln Ala Gly Ile 195 200 205 Ser Ser Phe Tyr Thr Lys Asp LeuIle Val Met Gly Ala Pro Gly Ser 210 215 220 Ser Tyr Trp Thr Gly Ser LeuPhe Val Tyr Asn Ile Thr Thr Asn Lys 225 230 235 240 Tyr Lys Ala Phe LeuAsp Lys Gln Asn Gln Val Lys Phe Gly Ser Tyr 245 250 255 Leu Gly Tyr SerVal Gly Ala Gly His Phe Arg Ser Gln His Thr Thr 260 265 270 Glu Val ValGly Gly Ala Pro Gln His Glu Gln Ile Gly Lys Ala Tyr 275 280 285 Ile PheSer Ile Asp Glu Lys Glu Leu Asn Ile Leu His Glu Met Lys 290 295 300 GlyLys Lys Leu Gly Ser Tyr Phe Gly Ala Ser Val Cys Ala Val Asp 305 310 315320 Leu Asn Ala Asp Gly Phe Ser Asp Leu Leu Val Gly Ala Pro Met Gln 325330 335 Ser Thr Ile Arg Glu Glu Gly Arg Val Phe Val Tyr Ile Asn Ser Gly340 345 350 Ser Gly Ala Val Met Asn Ala Met Glu Thr Asn Leu Val Gly SerAsp 355 360 365 Lys Tyr Ala Ala Arg Phe Gly Glu Ser Ile Val Asn Leu GlyAsp Ile 370 375 380 Asp Asn Asp Gly Phe Glu Asp Val Ala Ile Gly Ala ProGln Glu Asp 385 390 395 400 Asp Leu Gln Gly Ala Ile Tyr Ile Tyr Asn GlyArg Ala Asp Gly Ile 405 410 415 Ser Ser Thr Phe Ser Gln Arg Ile Glu GlyLeu Gln Ile Ser Lys Ser 420 425 430 Leu Ser Met Phe Gly Gln Ser Ile SerGly Gln Ile Asp Ala Asp Asn 435 440 445 Asn Gly Tyr Val Asp Val Ala ValGly Ala Phe Arg Ser Asp Ser Ala 450 455 460 Val Leu Leu Arg Thr Arg ProVal Val Ile Val Asp Ala Ser Leu Ser 465 470 475 480 His Pro Glu Ser ValAsn Arg Thr Lys Phe Asp Cys Val Glu Asn Gly 485 490 495 Trp Pro Ser ValCys Ile Asp Leu Thr Leu Cys Phe Ser Tyr Lys Gly 500 505 510 Lys Glu ValPro Gly Tyr Ile Val Leu Phe Tyr Asn Met Ser Leu Asp 515 520 525 Val AsnArg Lys Ala Glu Ser Pro Pro Arg Phe Tyr Phe Ser Ser Asn 530 535 540 GlyThr Ser Asp Val Ile Thr Gly Ser Ile Gln Val Ser Ser Arg Glu 545 550 555560 Ala Asn Cys Arg Thr His Gln Ala Phe Met Arg Lys Asp Val Arg Asp 565570 575 Ile Leu Thr Pro Ile Gln Ile Glu Ala Ala Tyr His Leu Gly Pro His580 585 590 Val Ile Ser Lys Arg Ser Thr Glu Glu Phe Pro Pro Leu Gln ProIle 595 600 605 Leu Gln Gln Lys Lys Glu Lys Asp Ile Met Lys Lys Thr IleAsn Phe 610 615 620 Ala Arg Phe Cys Ala His Glu Asn Cys Ser Ala Asp LeuGln Val Ser 625 630 635 640 Ala Lys Ile Gly Phe Leu Lys Pro His Glu AsnLys Thr Tyr Leu Ala 645 650 655 Val Gly Ser Met Lys Thr Leu Met Leu AsnVal Ser Leu Phe Asn Ala 660 665 670 Gly Asp Asp Ala Tyr Glu Thr Thr LeuHis Val Lys Leu Pro Val Gly 675 680 685 Leu Tyr Phe Ile Lys Ile Leu GluLeu Glu Glu Lys Gln Ile Asn Cys 690 695 700 Glu Val Thr Asp Asn Ser GlyVal Val Gln Leu Asp Cys Ser Ile Gly 705 710 715 720 Tyr Ile Tyr Val AspHis Leu Ser Arg Ile Asp Ile Ser Phe Leu Leu 725 730 735 Asp Val Ser SerLeu Ser Arg Ala Glu Glu Asp Leu Ser Ile Thr Val 740 745 750 His Ala ThrCys Glu Asn Glu Glu Glu Met Asp Asn Leu Lys His Ser 755 760 765 Arg ValThr Val Ala Ile Pro Leu Lys Tyr Glu Val Lys Leu Thr Val 770 775 780 HisGly Phe Val Asn Pro Thr Ser Phe Val Tyr Gly Ser Asn Asp Glu 785 790 795800 Asn Glu Pro Glu Thr Cys Met Val Glu Lys Met Asn Leu Thr Phe His 805810 815 Val Ile Asn Thr Gly Asn Ser Met Ala Pro Asn Val Ser Val Glu Ile820 825 830 Met Val Pro Asn Ser Phe Ser Pro Gln Thr Asp Lys Leu Phe AsnIle 835 840 845 Leu Asp Val Gln Thr Thr Thr Gly Glu Cys His Phe Glu AsnTyr Gln 850 855 860 Arg Val Cys Ala Leu Glu Gln Gln Lys Ser Ala Met GlnThr Leu Lys 865 870 875 880 Gly Ile Val Gln Phe Leu Ser Lys Thr Asp LysArg Leu Leu Tyr Cys 885 890 895 Ile Lys Ala Asp Pro His Cys Leu Asn PheLeu Cys Asn Phe Gly Lys 900 905 910 Met Glu Ser Gly Lys Glu Ala Ser ValHis Ile Gln Leu Glu Gly Arg 915 920 925 Pro Ser Ile Leu Glu Met Asp GluThr Ser Ala Leu Lys Phe Glu Ile 930 935 940 Arg Ala Thr Gly Phe Pro GluPro Asn Pro Arg Val Ile Glu Leu Asn 945 950 955 960 Lys Asp Glu Asn ValAla His Val Leu Leu Glu Gly Leu His His Gln 965 970 975 Arg Pro Lys ArgTyr Phe Thr Ile Val Ile Ile Ser Ser Ser Leu Leu 980 985 990 Leu Gly LeuIle Val Leu Leu Leu Ile Ser Tyr Val Met Trp Lys Ala 995 1000 1005 GlyPhe Phe Lys Arg Gln Tyr Lys Ser Ile Leu Gln Glu Glu Asn Arg 1010 10151020 Arg Asp Ser Trp Ser Tyr Ile Asn Ser Lys Ser Asn Asp Asp 1025 10301035 37 2742 DNA Homo sapiens CDS (114)...(2510) 37 agcccagagagaaagtctga cttgccccac agccagtgag tgactgcagc agcaccagaa 60 tctggtctgtttcctgtttg gctcttctac cactacggct tgggatctcg ggc atg 116 Met 1 gtg gctttg cca atg gtc ctt gtt ttg ctg ctg gtc ctg agc aga ggt 164 Val Ala LeuPro Met Val Leu Val Leu Leu Leu Val Leu Ser Arg Gly 5 10 15 gag agt gaattg gac gcc aag atc cca tcc aca ggg gat gcc aca gaa 212 Glu Ser Glu LeuAsp Ala Lys Ile Pro Ser Thr Gly Asp Ala Thr Glu 20 25 30 tgg cgg aat cctcac ctg tcc atg ctg ggg tcc tgc cag cca gcc ccc 260 Trp Arg Asn Pro HisLeu Ser Met Leu Gly Ser Cys Gln Pro Ala Pro 35 40 45 tcc tgc cag aag tgcatc ctc tca cac ccc agc tgt gca tgg tgc aag 308 Ser Cys Gln Lys Cys IleLeu Ser His Pro Ser Cys Ala Trp Cys Lys 50 55 60 65 caa ctg aac ttc accgcg tcg gga gag gcg gag gcg cgg cgc tgc gcc 356 Gln Leu Asn Phe Thr AlaSer Gly Glu Ala Glu Ala Arg Arg Cys Ala 70 75 80 cga cga gag gag ctg ctggct cga ggc tgc ccg ctg gag gag ctg gag 404 Arg Arg Glu Glu Leu Leu AlaArg Gly Cys Pro Leu Glu Glu Leu Glu 85 90 95 gag ccc cgc ggc cag cag gaggtg ctg cag gac cag ccg ctc agc cag 452 Glu Pro Arg Gly Gln Gln Glu ValLeu Gln Asp Gln Pro Leu Ser Gln 100 105 110 ggc gcc cgc gga gag ggt gccacc cag ctg gcg ccg cag cgg gtc cgg 500 Gly Ala Arg Gly Glu Gly Ala ThrGln Leu Ala Pro Gln Arg Val Arg 115 120 125 gtc acg ctg cgg cct ggg gagccc cag cag ctc cag gtc cgc ttc ctt 548 Val Thr Leu Arg Pro Gly Glu ProGln Gln Leu Gln Val Arg Phe Leu 130 135 140 145 cgt gct gag gga tac ccggtg gac ctg tac tac ctt atg gac ctg agc 596 Arg Ala Glu Gly Tyr Pro ValAsp Leu Tyr Tyr Leu Met Asp Leu Ser 150 155 160 tac tcc atg aag gac gacctg gaa cgc gtg cgc cag ctc ggg cac gct 644 Tyr Ser Met Lys Asp Asp LeuGlu Arg Val Arg Gln Leu Gly His Ala 165 170 175 ctg ctg gtc cgg ctg caggaa gtc acc cat tct gtg cgc att ggt ttt 692 Leu Leu Val Arg Leu Gln GluVal Thr His Ser Val Arg Ile Gly Phe 180 185 190 ggt tcc ttt gtg gac aaaacg gtg ctg ccc ttt gtg agc aca gta ccc 740 Gly Ser Phe Val Asp Lys ThrVal Leu Pro Phe Val Ser Thr Val Pro 195 200 205 tcc aaa ctg cgc cac ccctgc ccc acc cgg ctg gag cgc tgc cag tca 788 Ser Lys Leu Arg His Pro CysPro Thr Arg Leu Glu Arg Cys Gln Ser 210 215 220 225 cca ttc agc ttt caccat gtg ctg tcc ctg acg ggg gac gca caa gcc 836 Pro Phe Ser Phe His HisVal Leu Ser Leu Thr Gly Asp Ala Gln Ala 230 235 240 ttc gag cgg gag gtgggg cgc cag agt gtg tcc ggc aat ctg gac tcg 884 Phe Glu Arg Glu Val GlyArg Gln Ser Val Ser Gly Asn Leu Asp Ser 245 250 255 cct gaa ggt ggc ttcgat gcc att ctg cag gct gca ctc tgc cag gag 932 Pro Glu Gly Gly Phe AspAla Ile Leu Gln Ala Ala Leu Cys Gln Glu 260 265 270 cag att ggc tgg agaaat gtg tcc cgg ctg ctg gtg ttc act tca gac 980 Gln Ile Gly Trp Arg AsnVal Ser Arg Leu Leu Val Phe Thr Ser Asp 275 280 285 gac aca ttc cat acagct ggg gac ggg aag ttg ggc ggc att ttc atg 1028 Asp Thr Phe His Thr AlaGly Asp Gly Lys Leu Gly Gly Ile Phe Met 290 295 300 305 ccc agt gat gggcac tgc cac ttg gac agc aat ggc ctc tac agt cgc 1076 Pro Ser Asp Gly HisCys His Leu Asp Ser Asn Gly Leu Tyr Ser Arg 310 315 320 agc aca gag tttgac tac cct tct gtg ggt cag gta gcc cag gcc ctc 1124 Ser Thr Glu Phe AspTyr Pro Ser Val Gly Gln Val Ala Gln Ala Leu 325 330 335 tct gca gca aatatc cag ccc atc ttt gct gtc acc agt gcc gca ctg 1172 Ser Ala Ala Asn IleGln Pro Ile Phe Ala Val Thr Ser Ala Ala Leu 340 345 350 cct gtc tac caggag ctg agt aaa ctg att cct aag tct gca gtt ggg 1220 Pro Val Tyr Gln GluLeu Ser Lys Leu Ile Pro Lys Ser Ala Val Gly 355 360 365 gag ctg agt gaggac tcc agc aac gtg gta cag ctc atc atg gat gct 1268 Glu Leu Ser Glu AspSer Ser Asn Val Val Gln Leu Ile Met Asp Ala 370 375 380 385 tat aat agcctg tct tcc act gtg acc ctt gaa cac tct tca ctc cct 1316 Tyr Asn Ser LeuSer Ser Thr Val Thr Leu Glu His Ser Ser Leu Pro 390 395 400 cct ggg gtccac att tct tac gaa tcc cag tgt gag ggt cct gag aag 1364 Pro Gly Val HisIle Ser Tyr Glu Ser Gln Cys Glu Gly Pro Glu Lys 405 410 415 agg gag ggtaag gct gag gat cga gga cag tgc aac cac gtc cga atc 1412 Arg Glu Gly LysAla Glu Asp Arg Gly Gln Cys Asn His Val Arg Ile 420 425 430 aac cag acggtg act ttc tgg gtt tct ctc caa gcc acc cac tgc ctc 1460 Asn Gln Thr ValThr Phe Trp Val Ser Leu Gln Ala Thr His Cys Leu 435 440 445 cca gag ccccat ctc ctg agg ctc cgg gcc ctt ggc ttc tca gag gag 1508 Pro Glu Pro HisLeu Leu Arg Leu Arg Ala Leu Gly Phe Ser Glu Glu 450 455 460 465 ctg attgtg gag ttg cac acg ctg tgt gac tgt aat tgc agt gac acc 1556 Leu Ile ValGlu Leu His Thr Leu Cys Asp Cys Asn Cys Ser Asp Thr 470 475 480 cag ccccag gct ccc cac tgc agt gat ggc cag gga cac cta caa tgt 1604 Gln Pro GlnAla Pro His Cys Ser Asp Gly Gln Gly His Leu Gln Cys 485 490 495 ggt gtatgc agc tgt gcc cct ggc cgc cta ggt cgg ctc tgt gag tgc 1652 Gly Val CysSer Cys Ala Pro Gly Arg Leu Gly Arg Leu Cys Glu Cys 500 505 510 tct gtggca gag ctg tcc tcc cca gac ctg gaa tct ggg tgc cgg gct 1700 Ser Val AlaGlu Leu Ser Ser Pro Asp Leu Glu Ser Gly Cys Arg Ala 515 520 525 ccc aatggc aca ggg ccc ctg tgc agt gga aag ggt cac tgt caa tgt 1748 Pro Asn GlyThr Gly Pro Leu Cys Ser Gly Lys Gly His Cys Gln Cys 530 535 540 545 ggacgc tgc agc tgc agt gga cag agc tct ggg cat ctg tgc gag tgt 1796 Gly ArgCys Ser Cys Ser Gly Gln Ser Ser Gly His Leu Cys Glu Cys 550 555 560 gacgat gcc agc tgt gag cga cat gag ggc atc ctc tgc gga ggc ttt 1844 Asp AspAla Ser Cys Glu Arg His Glu Gly Ile Leu Cys Gly Gly Phe 565 570 575 ggtcgc tgc caa tgt gga gta tgt cac tgt cat gcc aac cgc acg ggc 1892 Gly ArgCys Gln Cys Gly Val Cys His Cys His Ala Asn Arg Thr Gly 580 585 590 agagca tgc gaa tgc agt ggg gac atg gac agt tgc atc agt ccc gag 1940 Arg AlaCys Glu Cys Ser Gly Asp Met Asp Ser Cys Ile Ser Pro Glu 595 600 605 ggaggg ctc tgc agt ggg cat gga cgc tgc aaa tgc aac cgc tgc cag 1988 Gly GlyLeu Cys Ser Gly His Gly Arg Cys Lys Cys Asn Arg Cys Gln 610 615 620 625tgc ttg gac ggc tac tat ggt gct cta tgc gac caa tgc cca ggc tgc 2036 CysLeu Asp Gly Tyr Tyr Gly Ala Leu Cys Asp Gln Cys Pro Gly Cys 630 635 640aag aca cca tgc gag aga cac cgg gac tgt gca gag tgt ggg gcc ttc 2084 LysThr Pro Cys Glu Arg His Arg Asp Cys Ala Glu Cys Gly Ala Phe 645 650 655agg act ggc cca ctg gcc acc aac tgc agt aca gct tgt gcc cat acc 2132 ArgThr Gly Pro Leu Ala Thr Asn Cys Ser Thr Ala Cys Ala His Thr 660 665 670aat gtg acc ctg gcc ttg gcc cct atc ttg gat gat ggc tgg tgc aaa 2180 AsnVal Thr Leu Ala Leu Ala Pro Ile Leu Asp Asp Gly Trp Cys Lys 675 680 685gag cgg acc ctg gac aac cag ctg ttc ttc ttc ttg gtg gag gat gac 2228 GluArg Thr Leu Asp Asn Gln Leu Phe Phe Phe Leu Val Glu Asp Asp 690 695 700705 gcc aga ggc acg gtc gtg ctc aga gtg aga ccc caa gaa aag gga gca 2276Ala Arg Gly Thr Val Val Leu Arg Val Arg Pro Gln Glu Lys Gly Ala 710 715720 gac cac acg cag gcc att gtg ctg ggc tgc gta ggg ggc atc gtg gca 2324Asp His Thr Gln Ala Ile Val Leu Gly Cys Val Gly Gly Ile Val Ala 725 730735 gtg ggg ctg ggg ctg gtc ctg gct tac cgg ctc tcg gtg gaa atc tat 2372Val Gly Leu Gly Leu Val Leu Ala Tyr Arg Leu Ser Val Glu Ile Tyr 740 745750 gac cgc cgg gaa tac agt cgc ttt gag aag gag cag caa caa ctc aac 2420Asp Arg Arg Glu Tyr Ser Arg Phe Glu Lys Glu Gln Gln Gln Leu Asn 755 760765 tgg aag cag gac agt aat cct ctc tac aaa agt gcc atc acg acc acc 2468Trp Lys Gln Asp Ser Asn Pro Leu Tyr Lys Ser Ala Ile Thr Thr Thr 770 775780 785 atc aat cct cgc ttt caa gag gca gac agt ccc act ctc tga 2510 IleAsn Pro Arg Phe Gln Glu Ala Asp Ser Pro Thr Leu * 790 795 aggagggagggacacttacc caaggctctt ctccttggag gacagtggga actggagggt 2570 gagaggaagggtgggtctgt aagaccttgg taggggacta attcactggc gaggtgcggc 2630 caccaccctacttcattttc agagtgacac ccaagagggc tgcttcccat gcctgcaacc 2690 ttgcatccatctgggctacc ccacccaagt atacaataaa gtcttacctc ag 2742 38 798 PRT Homosapiens 38 Met Val Ala Leu Pro Met Val Leu Val Leu Leu Leu Val Leu SerArg 1 5 10 15 Gly Glu Ser Glu Leu Asp Ala Lys Ile Pro Ser Thr Gly AspAla Thr 20 25 30 Glu Trp Arg Asn Pro His Leu Ser Met Leu Gly Ser Cys GlnPro Ala 35 40 45 Pro Ser Cys Gln Lys Cys Ile Leu Ser His Pro Ser Cys AlaTrp Cys 50 55 60 Lys Gln Leu Asn Phe Thr Ala Ser Gly Glu Ala Glu Ala ArgArg Cys 65 70 75 80 Ala Arg Arg Glu Glu Leu Leu Ala Arg Gly Cys Pro LeuGlu Glu Leu 85 90 95 Glu Glu Pro Arg Gly Gln Gln Glu Val Leu Gln Asp GlnPro Leu Ser 100 105 110 Gln Gly Ala Arg Gly Glu Gly Ala Thr Gln Leu AlaPro Gln Arg Val 115 120 125 Arg Val Thr Leu Arg Pro Gly Glu Pro Gln GlnLeu Gln Val Arg Phe 130 135 140 Leu Arg Ala Glu Gly Tyr Pro Val Asp LeuTyr Tyr Leu Met Asp Leu 145 150 155 160 Ser Tyr Ser Met Lys Asp Asp LeuGlu Arg Val Arg Gln Leu Gly His 165 170 175 Ala Leu Leu Val Arg Leu GlnGlu Val Thr His Ser Val Arg Ile Gly 180 185 190 Phe Gly Ser Phe Val AspLys Thr Val Leu Pro Phe Val Ser Thr Val 195 200 205 Pro Ser Lys Leu ArgHis Pro Cys Pro Thr Arg Leu Glu Arg Cys Gln 210 215 220 Ser Pro Phe SerPhe His His Val Leu Ser Leu Thr Gly Asp Ala Gln 225 230 235 240 Ala PheGlu Arg Glu Val Gly Arg Gln Ser Val Ser Gly Asn Leu Asp 245 250 255 SerPro Glu Gly Gly Phe Asp Ala Ile Leu Gln Ala Ala Leu Cys Gln 260 265 270Glu Gln Ile Gly Trp Arg Asn Val Ser Arg Leu Leu Val Phe Thr Ser 275 280285 Asp Asp Thr Phe His Thr Ala Gly Asp Gly Lys Leu Gly Gly Ile Phe 290295 300 Met Pro Ser Asp Gly His Cys His Leu Asp Ser Asn Gly Leu Tyr Ser305 310 315 320 Arg Ser Thr Glu Phe Asp Tyr Pro Ser Val Gly Gln Val AlaGln Ala 325 330 335 Leu Ser Ala Ala Asn Ile Gln Pro Ile Phe Ala Val ThrSer Ala Ala 340 345 350 Leu Pro Val Tyr Gln Glu Leu Ser Lys Leu Ile ProLys Ser Ala Val 355 360 365 Gly Glu Leu Ser Glu Asp Ser Ser Asn Val ValGln Leu Ile Met Asp 370 375 380 Ala Tyr Asn Ser Leu Ser Ser Thr Val ThrLeu Glu His Ser Ser Leu 385 390 395 400 Pro Pro Gly Val His Ile Ser TyrGlu Ser Gln Cys Glu Gly Pro Glu 405 410 415 Lys Arg Glu Gly Lys Ala GluAsp Arg Gly Gln Cys Asn His Val Arg 420 425 430 Ile Asn Gln Thr Val ThrPhe Trp Val Ser Leu Gln Ala Thr His Cys 435 440 445 Leu Pro Glu Pro HisLeu Leu Arg Leu Arg Ala Leu Gly Phe Ser Glu 450 455 460 Glu Leu Ile ValGlu Leu His Thr Leu Cys Asp Cys Asn Cys Ser Asp 465 470 475 480 Thr GlnPro Gln Ala Pro His Cys Ser Asp Gly Gln Gly His Leu Gln 485 490 495 CysGly Val Cys Ser Cys Ala Pro Gly Arg Leu Gly Arg Leu Cys Glu 500 505 510Cys Ser Val Ala Glu Leu Ser Ser Pro Asp Leu Glu Ser Gly Cys Arg 515 520525 Ala Pro Asn Gly Thr Gly Pro Leu Cys Ser Gly Lys Gly His Cys Gln 530535 540 Cys Gly Arg Cys Ser Cys Ser Gly Gln Ser Ser Gly His Leu Cys Glu545 550 555 560 Cys Asp Asp Ala Ser Cys Glu Arg His Glu Gly Ile Leu CysGly Gly 565 570 575 Phe Gly Arg Cys Gln Cys Gly Val Cys His Cys His AlaAsn Arg Thr 580 585 590 Gly Arg Ala Cys Glu Cys Ser Gly Asp Met Asp SerCys Ile Ser Pro 595 600 605 Glu Gly Gly Leu Cys Ser Gly His Gly Arg CysLys Cys Asn Arg Cys 610 615 620 Gln Cys Leu Asp Gly Tyr Tyr Gly Ala LeuCys Asp Gln Cys Pro Gly 625 630 635 640 Cys Lys Thr Pro Cys Glu Arg HisArg Asp Cys Ala Glu Cys Gly Ala 645 650 655 Phe Arg Thr Gly Pro Leu AlaThr Asn Cys Ser Thr Ala Cys Ala His 660 665 670 Thr Asn Val Thr Leu AlaLeu Ala Pro Ile Leu Asp Asp Gly Trp Cys 675 680 685 Lys Glu Arg Thr LeuAsp Asn Gln Leu Phe Phe Phe Leu Val Glu Asp 690 695 700 Asp Ala Arg GlyThr Val Val Leu Arg Val Arg Pro Gln Glu Lys Gly 705 710 715 720 Ala AspHis Thr Gln Ala Ile Val Leu Gly Cys Val Gly Gly Ile Val 725 730 735 AlaVal Gly Leu Gly Leu Val Leu Ala Tyr Arg Leu Ser Val Glu Ile 740 745 750Tyr Asp Arg Arg Glu Tyr Ser Arg Phe Glu Lys Glu Gln Gln Gln Leu 755 760765 Asn Trp Lys Gln Asp Ser Asn Pro Leu Tyr Lys Ser Ala Ile Thr Thr 770775 780 Thr Ile Asn Pro Arg Phe Gln Glu Ala Asp Ser Pro Thr Leu 785 790795 39 30 PRT Unknown acidic peptide 39 Ala Gln Leu Glu Lys Glu Leu GlnAla Leu Glu Lys Glu Asn Ala Gln 1 5 10 15 Leu Glu Trp Glu Leu Gln AlaLeu Glu Lys Glu Leu Ala Gln 20 25 30 40 30 PRT Unknown basic peptide 40Ala Gln Leu Lys Lys Lys Leu Gln Ala Leu Lys Lys Lys Asn Ala Gln 1 5 1015 Leu Lys Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala Gln 20 25 30 41 6PRT Artificial Sequence amino acid linker sequence 41 Gly Gly Ser ThrGly Gly 1 5 42 31 DNA Artificial Sequence Synthetic oligonucleotide 42gcactagtcc accatgggcc cttggagccg c 31 43 26 DNA Artificial SequenceSynthetic oligonucleotide 43 ccctcgagag gctgtgcctt cctaca 26 44 23 DNAArtificial Sequence Synthetic oligonucleotide 44 atctcgagcc caaatcttgtgac 23 45 28 DNA Artificial Sequence Synthetic oligonucleotide 45tagcggccgc tcatttaccc ggagacag 28 46 21 DNA Artificial SequenceSynthetic oligonucleotide 46 atggactgga cctggagcat c 21 47 20 DNAArtificial Sequence Synthetic oligonucleotide 47 atggaattgg ggctgagctg20 48 20 DNA Artificial Sequence Synthetic oligonucleotide 48 atggagtttggrctgagctg 20 49 21 DNA Artificial Sequence Synthetic oligonucleotide 49atgaaacacc tgtggttctt c 21 50 20 DNA Artificial Sequence Syntheticoligonucleotide 50 atggggtcaa ccgccatcct 20 51 21 DNA ArtificialSequence Synthetic oligonucleotide 51 tgccaggggg aagaccgatg g 21 52 33DNA Artificial Sequence Synthetic oligonucleotide 52 ttcttggtggcagcagccac aggtgcccac tcc 33 53 30 DNA Artificial Sequence Syntheticoligonucleotide 53 ctgctgacca tcccttcatg ggtcttgtcc 30 54 33 DNAArtificial Sequence Synthetic oligonucleotide 54 ttccttgttg ctattttaaaaggtgtccag tgt 33 55 27 DNA Artificial Sequence Syntheticoligonucleotide 55 gtggcagctc ccagatgggt cctgtcc 27 56 33 DNA ArtificialSequence Synthetic oligonucleotide 56 ctcctcctga ctgttctcca aggagtctgttcc 33 57 30 DNA Artificial Sequence Synthetic oligonucleotide 57gtgctgggcc tcccatgggg tgtcctgtca 30 58 30 DNA Artificial SequenceSynthetic oligonucleotide 58 ttggtggcag cagcaacagg tgcccactcc 30 59 26DNA Artificial Sequence Synthetic oligonucleotide 59 gaaggtgtgcacgccgctgg tcagag 26 60 66 DNA Artificial Sequence Syntheticoligonucleotide 60 gtgcaattgg tgcagtctgg agcagaggtg aaaaagcccggggagtctct gaaaatctcc 60 tgtaag 66 61 30 DNA Artificial SequenceSynthetic oligonucleotide 61 gtgcaattgg tggagtctgg gggaggcttg 30 62 32DNA Artificial Sequence Synthetic oligonucleotide 62 ctcgctgagctgacggtgac cagggttccc tg 32 63 57 DNA Artificial Sequence Syntheticoligonucleotide 63 ccggaattcc tcaccatgga aaccccagcg cagcttctcttcctcctgct actctgg 57 64 28 DNA Artificial Sequence Syntheticoligonucleotide 64 acccgtacgt ttgatctcca ccttggtc 28 65 45 DNAArtificial Sequence Synthetic oligonucleotide 65 ttacccaatt gtgtcctgtcccaggtgcag ctgcaggagt cgggc 45 66 40 DNA Artificial Sequence Syntheticoligonucleotide 66 tggaggctga gctgacggtg accgtggtcc cttggcccca 40 67 38DNA Artificial Sequence Synthetic oligonucleotide 67 ttcccagggtcccgttccga catccagatg acccagtc 38 68 31 DNA Artificial SequenceSynthetic oligonucleotide 68 agccaccgta cgtttgatat ccactctggt c 31 69 26DNA Artificial Sequence Synthetic oligonucleotide 69 tcggatccgctctggagaag gaggag 26 70 26 DNA Artificial Sequence Syntheticoligonucleotide 70 gcgaattcaa gggcgtctcc aaccgt 26

What is claimed is:
 1. An antibody or antigen-binding fragment thereofwhich binds an activated αE integrin, wherein said antibody orantigen-binding fragment specifically binds an activation-inducedepitope on integrin αE chain (CD103).
 2. The antibody or antigen-bindingfragment of claim 1 wherein said activation-induced epitope is inducedby activation with a divalent cation.
 3. The antibody or antigen-bindingfragment of claim 1 wherein said activation-induced epitope is in the Idomain of integrin αE chain (CD103).
 4. The antibody or antigen-bindingfragment of claim 1 wherein said antibody or antigen-binding fragment:a) inhibits binding of ligand to said αE integrin; b) inhibits αEintegrin-mediated adhesion of a cell expressing said αE integrin toepithelial cells or endothelial cells; or c) competitively inhibitsbinding of monoclonal antibody 3G6 to said αE integrin.
 5. The antibodyor antigen-binding fragment of claim 4 wherein said αE integrin is αEβ7integrin.
 6. The antibody or antigen-binding fragment of claim 4 whereinsaid antibody or antigen-binding fragment inhibits binding of ligand tosaid αE integrin, and said αE integrin is αEβ7 integrin and said ligandis E-cadherin.
 7. The antibody or antigen-binding fragment of claim 1wherein said antibody or antigen-binding fragment is selected from thegroup consisting of: a) a human antibody or an antigen-binding fragmentof a human antibody; b) a humanized antibody or an antigen-bindingfragment of a humanized antibody; and c) a chimeric antibody or anantigen-binding fragment of a chimeric antibody.
 8. An antibody orantigen-binding fragment thereof which binds an αE integrin and inhibitsbinding of ligand to said αE integrin, wherein said antibody orantigen-binding fragment comprises at least one heavy chaincomplementarity determining region (HCDR1, HCDR2 and/or HCDR3)comprising an amino acid sequence selected from the group consisting of:HCDR1 SEQ ID NO: 5 or SEQ ID NO: 5 wherein one or two amino acids areconservatively substituted; HCDR2 SEQ ID NO: 6 or SEQ ID NO: 6 whereinone or two amino acids are conservatively substituted; and HCDR3 SEQ IDNO: 7 or SEQ ID NO: 7 wherein one or two amino acids are conservativelysubstituted; and at least one light chain complementarity determiningregion (LCDR1, LCDR2 and/or LCDR3) comprising an amino acid sequenceselected from the group consisting of: LCDR1 SEQ ID NO: 10 or SEQ ID NO:10 wherein one or two amino acids are conservatively substituted; LCDR2SEQ ID NO: 11 or SEQ ID NO: 11 wherein one or two amino acids areconservatively substituted; and LCDR3 SEQ ID NO: 12 or SEQ ID NO: 12wherein one or two amino acids are conservatively substituted.
 9. Theantibody or antigen-binding fragment of claim 8 wherein said antibody orantigen-binding fragment selectively binds an activation-induced epitopeon integrin αE chain (CD103).
 10. An immunoglobulin heavy chain orantigen-binding portion thereof comprising three heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3) comprisingthe following amino acid sequences: HCDR1 SEQ ID NO: 5 or SEQ ID NO: 5wherein one or two amino acids are conservatively substituted; HCDR2 SEQID NO: 6 or SEQ ID NO: 6 wherein one or two amino acids areconservatively substituted; and HCDR3 SEQ ID NO: 7 or SEQ ID NO: 7wherein one or two amino acids are conservatively substituted, whereinan antibody comprising said heavy chain or antigen-binding portionthereof and a complementary light chain or antigen-binding portion of acomplementary light chain binds an αE integrin.
 11. An immunoglobulinlight chain or antigen-binding portion thereof comprising three lightchain complementarity determining regions (LCDR1, LCDR2 and LCDR3)comprising the following amino acid sequences: LCDR1 SEQ ID NO: 10 orSEQ ID NO: 10 wherein one or two amino acids are conservativelysubstituted; LCDR2 SEQ ID NO: 11 or SEQ ID NO: 11 wherein one or twoamino acids are conservatively substituted; and LCDR3 SEQ ID NO: 12 orSEQ ID NO: 12 wherein one or two amino acids are conservativelysubstituted, wherein an antibody comprising said light chain orantigen-binding portion thereof and a complementary heavy chain orantigen-binding portion of a complementary heavy chain binds an αEintegrin.
 12. An antibody or antigen-binding fragment thereof whichbinds an αE integrin and inhibits binding of ligand to said αE integrin,wherein a) said antibody or antigen-binding fragment comprises at leastone heavy chain complementarity determining region (HCDR1, HCDR2 and/orHCDR3) comprising an amino acid sequence selected from the groupconsisting of: HCDR1 SEQ ID NO: 15 or SEQ ID NO: 15 wherein one or twoamino acids are conservatively substituted; HCDR2 SEQ ID NO: 16 or SEQID NO: 16 wherein one or two amino acids are conservatively substituted;and HCDR3 SEQ ID NO: 17 or SEQ ID NO: 17 wherein one or two amino acidsare conservatively substituted; and at least one light chaincomplementarity determining region (LCDR1, LCDR2 and/or LCDR3)comprising an amino acid sequence selected from the group consisting of:LCDR1 SEQ ID NO: 20 or SEQ ID NO: 20 wherein one or two amino acids areconservatively substituted; LCDR2 SEQ ID NO: 21 or SEQ ID NO: 21 whereinone or two amino acids are conservatively substituted; and LCDR3 SEQ IDNO: 22 or SEQ ID NO: 22 wherein one or two amino acids areconservatively substituted; or b) said antibody or antigen-bindingfragment comprises at least one heavy chain complementarity determiningregion (HCDR1, HCDR2 and/or HCDR3) comprising an amino acid sequenceselected from the following: HCDR1 SEQ ID NO: 25 or SEQ ID NO: 25 whereone or two amino acids are conservatively substituted; HCDR2 SEQ ID NO:26 or SEQ ID NO: 26 where one or two amino acids are conservativelysubstituted; and HCDR3 SEQ ID NO: 27 or SEQ ID NO: 27 where one or twoamino acids are conservatively substituted; and at least one light chaincomplementarity determining region (LCDR1, LCDR2 and/or LCDR3)comprising an amino acid sequence selected from the group consisting of:LCDR1 SEQ ID NO: 30 or SEQ ID NO: 30 wherein one or two amino acids areconservatively substituted; LCDR2 SEQ ID NO: 31 or SEQ ID NO: 31 whereinone or two amino acids are conservatively substituted; and LCDR3 SEQ IDNO: 32 or SEQ ID NO: 32 wherein one or two amino acids areconservatively substituted.
 13. The antibody or antigen-binding fragmentof claim 12 wherein said antibody has the epitopic specificity ofmonoclonal antibody 5E4 or monoclonal antibody 8D5.
 14. Animmunoglobulin heavy chain or antigen-binding portion thereofcomprising: a) three heavy chain complementarity determining regions(HCDR1, HCDR2 and HCDR3) comprising the following amino acid sequences:HCDR1 SEQ ID NO: 15 or SEQ ID NO: 15 wherein one or two amino acids areconservatively substituted; HCDR2 SEQ ID NO: 16 or SEQ ID NO: 16 whereinone or two amino acids are conservatively substituted; and HCDR3 SEQ IDNO: 17 or SEQ ID NO: 17 wherein one or two amino acids areconservatively substituted; or b) three heavy chain complementaritydetermining regions (HCDR1, HCDR2 and HCDR3) comprising the followingamino acid sequences: HCDR1 SEQ ID NO: 25 or SEQ ID NO: 25 wherein oneor two amino acids are conservatively substituted; HCDR2 SEQ ID NO: 26or SEQ ID NO: 26 wherein one or two amino acids are conservativelysubstituted; and HCDR3 SEQ ID NO: 27 or SEQ ID NO: 27 wherein one or twoamino acids are conservatively substituted, wherein an antibodycomprising said heavy chain or antigen-binding portion thereof and acomplementary light chain or antigen-binding portion of a complementarylight chain binds an αE integrin.
 15. An isolated and/or recombinantnucleic acid comprising a nucleotide sequence that encodes theimmunoglobulin heavy chain or antigen-binding portion thereof of claim10 or
 14. 16. An expression construct comprising a recombinant nucleicacid comprising a nucleotide sequence that encodes the immunoglobulinheavy chain or antigen-binding portion thereof of claim 10 or
 14. 17. Ahost cell comprising a recombinant nucleic acid encoding theimmunoglobulin heavy chain or antigen-binding portion thereof of claim10 or
 14. 18. An immunoglobulin light chain or antigen-binding portionthereof comprising: a) three light chain complementarity determiningregions (LCDR1, LCDR2 and LCDR3) comprising the following amino acidsequences: LCDR1 SEQ ID NO: 20 or SEQ ID NO: 20 wherein one or two aminoacids are conservatively substituted; LCDR2 SEQ ID NO: 21 or SEQ ID NO:21 wherein one or two amino acids are conservatively substituted; andLCDR3 SEQ ID NO: 22 or SEQ ID NO: 22 wherein one or two amino acids areconservatively substituted; or b) three light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) comprising the followingamino acid sequences: LCDR1 SEQ ID NO: 30 or SEQ ID NO: 30 wherein oneor two amino acids are conservatively substituted; LCDR2 SEQ ID NO: 31or SEQ ID NO: 31 wherein one or two amino acids are conservativelysubstituted; and LCDR3 SEQ ID NO: 32 or SEQ ID NO: 32 wherein one or twoamino acids are conservatively substituted, wherein an antibodycomprising said light chain or antigen-binding portion thereof and acomplementary heavy chain or antigen-binding portion of a complementaryheavy chain binds an αE integrin.
 19. An isolated and/or recombinantnucleic acid comprising a nucleotide sequence that encodes theimmunoglobulin light chain or antigen-binding portion thereof of Claim11 or
 18. 20. An expression construct comprising a recombinant nucleicacid comprising a nucleotide sequence that encodes the immunoglobulinlight chain or antigen-binding portion thereof of claim 11 or
 18. 21. Ahost cell comprising a recombinant nucleic acid encoding theimmunoglobulin light chain or antigen-binding portion thereof of claim11 or 18
 22. An isolated cell that produces the antibody orantigen-binding fragment of claim 1, 8 or
 12. 23. An antibody orantigen-binding fragment thereof, wherein said antibody or fragment isselected from the group consisting of: a) the antibody produced byhybridoma 3G6 (ATCC Accession No. PTA-4201) or an antigen-bindingfragment thereof; b) the antibody produced by hybridoma 5E4 (ATCCAccession No. PTA-4202) or an antigen-binding fragment thereof; c) theantibody produced by hybridoma 8D5 (ATCC Accession No. PTA-4203) or anantigen-binding fragment thereof; d) the antibody produced by CHO 3G6C1.2D6 (ATCC Accession No. PTA-4204) or an antigen-binding fragmentthereof; and e) the antibody produced by CHO 5G4 A1.2C12 (ATCC AccessionNo. PTA-4205) or an antigen-binding fragment thereof.
 24. Hybridoma 3G6(ATCC Accession No. PTA-4201), Hybridoma 5E4 (ATCC Accession No.PTA-4202), Hybridoma 8D5 (ATCC Accession No. PTA-4203), CHO 3G6 C1.2D6(ATCC Accession No. PTA-4204) or CHO 5G4 A1.2C12 (ATCC Accession No.PTA-4205).
 25. A composition comprising the antibody or antigen-bindingfragment of claim 1, 8 or 12 and a physiologically acceptable carrier.26. A method for treating a subject having an inflammatory disorder,comprising administering to said subject an effective amount of anantibody or antigen-binding fragment thereof which specifically binds anactivated αE integrin, wherein said antibody or antigen-binding fragmentbinds an activation-induced epitope on integrin αE chain (CD103). 27.The method of claim 30 wherein the inflammatory disorder is aninflammatory bowel disease.
 28. A method for treating a subject havingan inflammatory disorder, comprising administering to said subject aneffective amount of an antibody or antigen-binding fragment thereofwhich binds an αE integrin and inhibits binding of ligand to said αEintegrin, wherein: a) said antibody or antigen-binding fragmentcomprises at least one heavy chain complementarity determining region(HCDR1, HCDR2 and/or HCDR3) comprising an amino acid sequence selectedfrom the group consisting of: HCDR1 SEQ ID NO: 5 or SEQ ID NO: 5 whereinone or two amino acids are conservatively substituted; HCDR2 SEQ ID NO:6 or SEQ ID NO: 6 wherein one or two amino acids are conservativelysubstituted; and HCDR3 SEQ ID NO: 7 or SEQ ID NO: 7 wherein one or twoamino acids are conservatively substituted; and at least one light chaincomplementarity determining region (LCDR1, LCDR2 and/or LCDR3)comprising an amino acid sequence selected from the group consisting of:LCDR1 SEQ ID NO: 10 or SEQ ID NO: 10 where one or two amino acids areconservatively substituted; LCDR2 SEQ ID NO: 11 or SEQ ID NO: 11 whereone or two amino acids are conservatively substituted; and LCDR3 SEQ IDNO: 12 or SEQ ID NO: 12 where one or two amino acids are conservativelysubstituted; or b) said antibody or antigen-binding fragment comprisesat least one heavy chain complementarity determining region (HCDR1,HCDR2 and/or HCDR3) comprising an amino acid sequence selected from thegroup consisting of: HCDR1 SEQ ID NO: 15 or SEQ ID NO: 15 wherein one ortwo amino acids are conservatively substituted; HCDR2 SEQ ID NO: 16 orSEQ ID NO: 16 wherein one or two amino acids are conservativelysubstituted; and HCDR3 SEQ ID NO: 17 or SEQ ID NO: 17 wherein one or twoamino acids are conservatively substituted; and at least one light chaincomplementarity determining region (LCDR1, LCDR2 and/or LCDR3)comprising an amino acid sequence selected from the following: LCDR1 SEQID NO: 20 or SEQ ID NO: 20 wherein one or two amino acids areconservatively substituted; LCDR2 SEQ ID NO: 21 or SEQ ID NO: 21 whereinone or two amino acids are conservatively substituted; and LCDR3 SEQ IDNO: 22 or SEQ ID NO: 22 wherein one or two amino acids areconservatively substituted; or c) said antibody or antigen-bindingfragment comprises at least one heavy chain complementarity determiningregion (HCDR1, HCDR2 and/or HCDR3) comprising an amino acid sequenceselected from the group consisting of: HCDR1 SEQ ID NO: 25 or SEQ ID NO:25 wherein one or two amino acids are conservatively substituted; HCDR2SEQ ID NO: 26 or SEQ ID NO: 26 wherein one or two amino acids areconservatively substituted; and HCDR3 SEQ ID NO: 27 or SEQ ID NO: 27wherein one or two amino acids are conservatively substituted; and atleast one light chain complementarity determining region (LCDR1, LCDR2and/or LCDR3) comprising an amino acid sequence selected from the groupconsisting of: LCDR1 SEQ ID NO: 30 or SEQ ID NO: 30 wherein one or twoamino acids are conservatively substituted; LCDR2 SEQ ID NO: 31 or SEQID NO: 31 wherein one or two amino acids are conservatively substituted;and LCDR3 SEQ ID NO: 32 or SEQ ID NO: 32 wherein one or two amino acidsare conservatively substituted.
 29. A method for detecting an activatedαE integrin comprising contacting a composition comprising an αEintegrin with an antibody or antigen-binding fragment thereof whichbinds an activation-induced epitope on integrin αE chain (CD103) anddetecting formation of a complex between said antibody orantigen-binding fragment and said activated αE integrin.